Molecular Diagnostics System and Methods

ABSTRACT

The present invention relates to methods for the extraction of nucleic acids from cells, the amplification of segments of nucleic acid and the detection of nucleic acids, all in a convenient and portable manner. In one embodiment, a sample comprising cells containing nucleic acid is exposed to an aqueous mixture comprising a lytic reagent and one or more beads capable of binding the nucleic acid released from said cells to form a nucleic acid-bead complex. The nucleic acid-bead complex is passed through an immiscible liquid layer to separate the nucleic acid from the aqueous mixture. The one or more beads are magnetic, and the nucleic acid-bead complex is passed through and separated from the immiscible liquid layer with an applied magnetic field. The invention is particularly suited for use in point-of-care medical diagnostics testing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/308,307, filed on May 14, 2010, which is a U.S. National StageApplication of PCT/US2005/046772, filed on Dec. 21, 2005, which claimsthe benefit of U.S. Provisional Application No. 60/638,177 filed Dec.23, 2004. Each of these applications is incorporated herein by referencein its entirety.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submitted inpaper copy and computer readable form (CRF) and is hereby incorporatedby reference in its entirety. Said CRF copy, file“2010-03-17-215105-01708_ST25.txt” filed in U.S. application Ser. No.12/308,307, on May 14, 2010, is identical to the paper copy, except thatit lacks formatting.

FIELD OF THE INVENTION

The present invention relates to an integrated nucleic acid testcartridge capable of performing extraction, amplification and detectiontogether. It also relates to devices and methods for nucleic acidextraction alone, or extraction and amplification combined. Furthermore,it relates to devices and methods for amplification and detectioncombined. The cartridge may be equipped with a sensing means includingenabling optical and electrochemical detection methods and it may alsobe equipped with a wax or absorbent filter extraction feature toseparate target nucleic acid from the sample. The cartridge can performvarious methods of amplification including polymerase chain reaction,rolling circle amplification and strand displacement amplification. Thepresent invention also addresses novel amplification methods andreagents comprising single modified primers or pairs of modifiedprimers, depending on the selected amplification method. Furthermore thepresent invention provides for integrated electrophoretic separation forprimers from amplicons during a nucleic acid test.

BACKGROUND OF THE INVENTION General Background on the Value of NucleicAcid Testing

Applications of nucleic acid testing are broad. The majority of currentcommercial testing relates to infectious diseases including Chlamydia,gonorrhea, hepatitis and human immunodeficiency virus (HIV) viral load;genetic diseases including cystic fibrosis; coagulation and hematologyfactors including hemochromatosis; and cancer including genes for breastcancer. Other areas of interest include cardiovascular diseases and drugresistance screening, termed pharmacogenomics. The majority of testingcurrently occurs in centralized laboratories using non-portable andoperationally complex instruments. Presently, tests generally requirehighly skilled individuals to perform the assays. As a result, the timetaken between obtaining a sample suspected of containing a specificnucleic acid fragment and determining its presence or absence is oftenseveral hours and even days. However, as with other kinds of bloodtests, physicians and others often require results more quickly andobtainable in a convenient user-friendly format. Consequently, there isa need for a portable analysis system capable of performing nucleic acidtesting quickly and conveniently. A discussion of prior art relating tovarious aspects of nucleic acid testing is provided in the followingsections.

Methods to Characterize Genetic Information

The clinical manifestation of a particular genetic characteristic can bedifferent with different types or classes of genetic based diseases.This translates into different approaches to measure the geneticcharacteristic including SNP mutation detection, gene copy mutations andgene overexpression mutations. For example, some diseases such ashemochromatosis, cystic fibrosis or the oncogene p53, have one or a fewvery specific mutations which affect only a specific nucleotide.Considering hemochromatosis, there are two specific mutations. Theclinical manifestation of this disease is an accumulation of iron invarious tissues, which can be fatal if untreated. The most prevalentmutation is the G to A transition at nucleotide 845 in the gene, alsoknown as (C282Y). See OMIM: Online Mendelian Inheritance in Mandatabase, which can be found at the U.S. National Center for BiologicInformation internet site. The second most prevalent mutation in thesame hemochromatosis gene is a C to G transversion in exon 2, known asH63D. These are known as single nucleotide polymorphisms (SNPs). Asevery individual has two copies of each gene, the possible combinationsof these genes are two wild type (homozygous wild type), two mutatedgenes (homozygous mutant) or one wild type and one mutated gene(heterozygous). In the case of hemochromatosis, individuals who arehomozygous mutant exhibit the disease state, heterozygous individualscan be susceptible for some aspects of the disease as they accumulatehigher levels of iron than do homozygous wildtype individuals. Also, forthe purpose of determining if an individual is a carrier of the diseaseto their offspring, the ability to determine that an individual isheterozygous can be useful.

As a result, in testing for a genetic disease like hemochromatosis, itis useful to be able to have at least four analytical means or channelsfor detection. Here, one channel detects the presence of wild type C282,a second channel detects the presence of the mutant Y282 gene, a thirdchannel detects the presence of the wildtype H63 gene and the fourthchannel detects the presence of the mutant D63 gene. FIG. 12 provides atable of possible outcomes from a hemochromatosis test of this type andshows that it is possible to differentiate between homozygous orheterozygous, and that homozygous channels generate roughly twice thelevel of expression and thus signal in the test. Note that it is alsouseful to have one or more additional channels to use as positive andnegative controls.

Some genetic mutations include multiple copies of the gene being presentin the genome, causing a disease state in a patient. As an example theoncogene ZNF217 mapped within 20q13.2 has been found in multiple copiesin individuals with colon cancer (Rooney et al., 2004, J. Pathol. Vol204(3):282). Genetic triplication of the alpha-synuclein gene (SNCA) hasbeen reported to cause hereditary early-onset Parkinsonism with dementia(Chartier-Harlin et al., 2004, Lancet, vol 364(9440):1167). Yamashita etal., 2004, European Neurology, vol 52(2): 101, have found that there isan increase in adult-onset Type III spinal muscular atrophy related toincreased gene copies of the survival motor neuron (SMN2) gene. Thesegene copy mutations can be detected by using one or more required genes,such as the housekeeping genes (e.g. actin or glyceraldehyde 3-phosphatedehydrogenase). Overexpression mutations typically generate increasedlevels of mRNA and these can be detected.

Methods and Apparatuses for Extraction of Nucleic Acid

Nucleic acids found in cells can be deoxyribonucleic acid or ribonucleicacid and can be genomic DNA, extrachromosomal DNA (e.g. plasmids andepisomes), mitochondrial DNA, messenger RNA and transfer RNA. Nucleicacids can also be foreign to the host and contaminate a cell as aninfectious agent, e.g. bacteria, viruses, fungi or single celledorganisms and infecting multicellular organisms (parasites). Recently,detection and analysis of the presence of nucleic acids has becomeimportant for the identification of single nucleotide polymorphisms(SNPs), chromosomal rearrangements and the insertion of foreign genes.These include infectious viruses, e.g. HIV and other retroviruses,jumping genes, e.g. transposons, and the identification of nucleic acidsfrom recombinantly engineered organisms containing foreign genes, e.g.Roundup Ready™ plants.

The analysis of nucleic acids has a wide array of uses. For example, thepresence of a foreign agent can be used as a medical diagnostic tool.The identification of the genetic makeup of cancerous tissues can alsobe used as a medical diagnostic tool, confirming that a tissue iscancerous, and determining the aggressive nature of the canceroustissue. Chromosomal rearrangements, SNPs and abnormal variations in geneexpression can be used as a medical diagnostic for particular diseasestates. Further, genetic information can be used to ascertain theeffectiveness of particular pharmaceutical drugs, known as the field ofpharmacogenomics. Genetic variations between humans and between domesticanimals can also be ascertained by DNA analysis. This is used in fieldsincluding forensics, paternity testing and animal husbandry.

Methods of extracting nucleic acids from cells are well known to thoseskilled in the art. A cell wall can be weakened by a variety of methods,permitting the nucleic acids to extrude from the cell and permitting itsfurther purification and analysis. The specific method of nucleic acidextraction is dependent on the type of nucleic acid to be isolated, thetype of cell, and the specific application used to analyze the nucleicacid. Many methods of isolating DNA are known to those skilled in theart, see for example the general reference Sambrook and Russell, 2001,“Molecular Cloning: A Laboratory Manual”. For example, the prior artcontains examples of chemically-impregnated and dehydratedsolid-substrates for the extraction and isolation of DNA from bodilyfluids that employ lytic salts and detergents and which containadditional reagents for long-term storage of DNA samples e.g. U.S. Pat.No. 5,807,527 detailing FTA paper and U.S. Pat. No. 6,168,922 detailingIsocard Paper. The prior art also contains examples of particleseparation methods, e.g. U.S. RE 37,891.

Methods of isolating RNA, particularly messenger RNA (mRNA) are wellknown to those skilled in the art. Typically, cell disruption isperformed in the presence of strong protein denaturing solutions, whichinactivate RNAses during the RNA isolation procedure. RNA is thenisolated using differential ethanol precipitation with centrifugation.As is well known, RNA is extremely labile and is sensitive to alkalineconditions, as well as RNAses, which degrade RNA. RNAses are ubiquitouswithin the environment and it has been found that they are difficult toremove from solutions and containers used to isolate RNA.

Methods and Apparatuses for Amplification of Nucleic Acid

Polymerase Chain Reaction (PCR) is inhibited by a number of proteins andother contaminants that follow through during the standard methods ofpurification of genomic DNA from a number of types of tissue samples. Itis known that additional steps of organic extraction with phenol,chloroform and ether or column chromatography or gradient CsC 1ultracentrifugation can be performed to remove PCR inhibitors in genomicDNA samples from blood. However, these steps add time, complexity andcost. This complexity limits incorporation into a simple disposablecartridge useful for nucleic acid analysis. Therefore, the developmentof new simple methods to overcome inhibitors found in nucleic acidsamples used for nucleic acid amplification processes is desirable.

Nucleic acid hybridization is used to detect discernible characteristicsabout target nucleic acid molecules. Techniques like the “Southernanalysis” are well known to those skilled in the art. Target nucleicacids are electrophoretically separated then bound to a membrane.Labeled probe molecules are then permitted to hybridize to the nucleicacids bound to the membrane using techniques well known in the art. Thismethod is limited, because the sensitivity of detection is dependent onthe amount of target material and the specific activity of the probe. Asthe probe's specific activity may be fixed, to improve the sensitivityof these assays, methods of amplifying nucleic acids are employed. Twobasic strategies are employed for nucleic acid amplification techniques;either the number of target copies is amplified, which in turn increasesthe sensitivity of detection, or the presence of the nucleic acid isused to increase a signal generated for detection. Examples of the firstapproach are polymerase chain reaction (PCR), rolling circle (see U.S.Pat. No. 5,854,033), and nucleic acid system based amplification(NASBA). Examples of the second include, cycling probe reaction, termedCPR (see U.S. Pat. No. 4,876,187 and U.S. Pat. No. 5,660,988) and SNPaseassays, e.g. the Mismatch Identification DNA Analysis System (see U.S.Pat. No. 5,656,430 and U.S. Pat. No. 5,763,178).

The PCR reaction is well known to those skilled in the art and wasoriginally described in U.S. Pat. No. 4,683,195. The process involvesdenaturing nucleic acid, a hybridization step and an extension step inrepeated cycles and is performed by varying the temperature of thenucleic acid sample and reagents. This process of subjecting the samplesto different temperatures can be effected by placing tubes intodifferent temperature water baths, or by using peltier-based devicescapable of generating heating or cooling, dependent on the direction ofthe electrical current as described in U.S. Pat. No. 5,333,675 and U.S.Pat. No. 5,656,493. Many commercial temperature cycling devices areavailable, sold for example by Perkin Elmer, Applied Biosystems andEppendorf. As these devices are generally large and heavy they are notgenerally amenable to use in non-laboratory environments, e.g. at thepoint-of-care.

A microfabricated device for performing the polymerase chain reaction isdescribed in U.S. Pat. No. 5,639,423 though it is silent on providing anintegrated means for extracting nucleic acids. A device for performingthe polymerase chain reaction is described in U.S. Pat. No. 5,645,801which has an amplification chamber that can be mated in a sealablemanner to a chamber for detection. U.S. Pat. No. 5,939,312 describes aminiaturized multi-chamber polymerase chain reaction device. U.S. Pat.No. 6,054,277 describes a silicon-based miniaturized genetic testingplatform for amplification and detection. A polymer-based heatingcomponent for amplification reactions is described in U.S. Pat. No.6,436,355. U.S. Pat. No. 6,303,288 describes an amplification anddetection system with a rupturable pouch containing reagents foramplification. U.S. Pat. No. 6,372,484 describes an apparatus forperforming the polymerase chain reaction and subsequent capillaryelectrophoretic separation and detection in an integrated device.

There are several nucleic acid amplification technologies that differfrom the PCR reaction in that the reaction is run at a singletemperature. These isothermal methods include the cycling probereaction, strand displacement, Invader™, SNPase, rolling circle reactionand NASBA. U.S. Pat. No. 6,379,929 describes a device for performing anisothermal nucleic acid amplification reaction.

More recently, a strategy for performing the polymerase chain reactionisothermally has been described by Vincent et al., 2004, EMBO Reports,vol 5(8), see also US Application 20040058378. A DNA helicase enzyme isused to overcome the limitations of heating a sample to perform PCR DNAamplification.

Enzymes Used for the Polymerase Chain Reaction (PCR)

The polymerase chain reaction (PCR) is based on the ability of a DNApolymerase enzyme to exhibit several core features, which include itsability to use a primer sequence with a 3′-hydroxyl group and a DNAtemplate sequence and to extend a newly synthesized strand of DNA usingthe template strand, all well known to those skilled in the art. Inaddition, DNA polymerases used in the PCR reaction must be able towithstand high temperatures (e.g. 90 to 99° C.) used to denature doublestranded DNA templates, as well as be inactive at lower temperatures(e.g. 40 to 60° C.) at which DNA primers hybridize to the DNA template.Further, to have optimal DNA synthesis at a temperature near to thehybridization temperature (e.g. 60 to 80° C.).

In addition to these core characteristics, DNA polymerases also exhibitproofreading capabilities, which are due to the 3′-5′ exonucleaseactivity inherent in most DNA polymerases. For the purpose of singlenucleotide polymorphism (SNP) detection based on differential primerextension using PCR (also called 3′-allele specific primer extension),it is a disadvantage to use an enzyme that exhibits a 3′-5′ exonucleaseactivity, as the terminal 3′ nucleotide can be excised from a standardnucleic acid primer, permitting synthesis of both alleles.

Zhang et al., (2003, Laboratory Investigation, vol 83(8): 1147) describethe use of a terminal phosphorothioate bond to overcome the limitationsof DNA polymerases used for 3′-5′ exonuclease activity. Thephosphorothioate bond is not cleaved by 3′-5′ exonucleases. Thisprevents DNA polymerases with 3′-5′ exonuclease activities from removingthe terminal mismatch and proceeding with DNA elongation, alleviatingthe lack of discrimination observed with normal DNA.

Another characteristic of DNA polymerases is their elongation rate.Takagi et al., (1997, Applied and Environmental Microbiology, vol63(11): 4504) teach that Pyrococcus sp. Strain KOD1 (now Thermococcuskodakaraensis KOD1), Pyrococcus furiosus, Deep Vent (New EnglandBiolabs, Beverly, Mass.), and Thermus aquaticus have elongation rates of106 to 138,25,23 and 61 bases/second, respectively. The processivityrates of these enzymes are also described, and behave similarly to theelongation rates. Clearly, Thermococcus kodakaerensis KOD1 has muchhigher elongation and processivity rates compared to the otherwell-known enzymes, which would make this enzyme beneficial inapplications where sensitivity and speed are an issue. Further,Thermococcus kodakaerensis KOD1 possesses an exonuclease activity whichwould be detrimental for use in a 3′-allele specific primer extensionassay used for SNP analysis.

Design of Synthetic Oligonucleotides

Regarding the design of synthetic oligonucleotides for use inamplification reactions, Rychlik et al. (1989, Nucleic Acids Research,vol 17(21):8543-8551) and Rychlik (1995, Molecular Biotechnology, vol 3:129-134), describe selection criteria and computer programs to designprobes and primers, including primers for in vitro amplification of DNA.Both teach that primers should not generate secondary structure orexhibit self-hybridization.

PCR primers designed as molecular beacons (Bonnet et al., 1999. Proc.Natl. Acad. Sci. USA, vol 96: 6171-6176) have a short region at both the5′ and 3′ ends which are complementary generating what is known ashairpin loop structures, to quench the fluorescent signal by placing thedonor and quencher molecules in close physical proximity to each other.After polymerization and incorporation into a newly synthesized doublestranded molecule, the donor and quencher molecules are physicallydistant to each other, permitting the generation of a fluorescentsignal. The region of complementarity is short and typically has onlyabout 5 nucleotides which are complementary, preferably generating ahairpin stem. Tsourkas et al., 2003, Nucleic Acids Research, vol 31(4):1319-1330, teaches that molecular beacons with longer stem lengths havean improved ability to discriminate between targets over a broader rangeof temperatures. However, this is accompanied by a decrease in the rateof molecular beacon-target hybridization. Molecular beacons with longerprobe lengths tend to have lower dissociation constants, increasedkinetic rate constants and decreased specificity. Therefore, havinglonger stem loops will have an impact on reducing the efficiency ofhybridization kinetics, which in turn will reduce the levels of PCRamplification. Therefore, PCR using a stem loop structure is generallyundesirable in the art. Kaboev et al., (2000, Nucleic Acids Research,vol 28(21):e94) teaches that designing a PCR primer with a stem loopstructure by adding additional sequences to the 5′-end of the primer,which are complementary to the 3′-end. This reference also teaches thatadding this secondary structure increases the specificity of the PCRreaction, though it does use a PCR primer that permits the generation ofsingle stranded tails. Further, Kaboev does not teach that thegeneration of the secondary structure prevents the hybridization of thesingle stranded regions to a capture moiety.

Detection Methods

Conventional detection methods for the final step in a nucleic acidanalysis are well known in the art and include sandwich-type capturemethods based on radioactivity, colorimetry, fluorescence, fluorescenceresonance energy transfer (FRET) and electrochemistry. For example,jointly owned U.S. Pat. No. 5,063,081 covers a sensor for nucleic aciddetection. The sensor has a permselective layer over an electrode and aproteinaceous patterned layer with an immobilized captureoligonucleotide. The oligonucleotide can be a polynucleotide, DNA, RNA,active fragments or subunits or single strands thereof. Coupling meansfor immobilizing nucleic acids are described along with methods where animmobilized nucleic acid probe binds to a complimentary target sequencein a sample. Detection is preferably electrochemical and is based on alabeled probe that also binds to a different region of the target.Alternatively, an immobilized antibody to the hybrid formed by a probeand polynucleotide sequence can be used along with DNA binding proteins.The '081 patent incorporates by reference the jointly owned U.S. Pat.No. 5,096,669 which covers a single-use cartridge for performing assaysin a sample using sensors. These sensors can be of the type described in'081.

Other divisional patents related to '081 include U.S. Pat. No. 5,200,051which covers a method of making a plurality of sensors with apermselective membrane coated with a ligand receptor that can be anucleic component. U.S. Pat. No. 5,554,339 covers microdispensing, wherea nucleic acid component is incorporated into a film-forming latex or aproteinaceous photoformable matrix for dispensing. U.S. Pat. No.5,466,575 covers methods for making sensors with the nucleic componentincorporated into a film-forming latex or a proteinaceous photoformablematrix. U.S. Pat. No. 5,837,466 covers methods for assaying a ligandusing the sensor components including nucleic components. For example, aquantitative oligonucleotide assay is described where the target bindsto a receptor on the sensor and is also bound by a labeled probe. Thelabel is capable of generating a signal that is detected by the sensor,e.g. an electrochemical sensor. U.S. Pat. No. 5,837,454 covers a methodof making a plurality of sensors with a permselective membrane coatedwith a ligand receptor that can be a nucleic component. Finally, jointlyowned U.S. Pat. No. 5,447,440 covers a coagulation affinity-based assayapplicable to nucleotides, oligonucleotides or polynucleotides. Thesejointly owned patents are incorporated herein by reference.

It is noteworthy that jointly owned U.S. Pat. No. 5,609,824 discloses athermostated chip for use within a disposable cartridge applicable tothermostating a sample, e.g. blood, to 37° C. Jointly owned U.S. Pat.No. 6,750,053 and pending US 20030170881 address functional fluidicelements of a disposable cartridge relevant to various tests includingDNA analyses. These additional jointly owned patents and applicationsare incorporated herein by reference. Several other patents addresselectrochemical detection of nucleic acids, for example U.S. Pat. No.4,840,893 discloses detection with an enzyme label that uses a mediator,e.g. ferrocene. U.S. Pat. No. 6,391,558 discloses single stranded DNA onthe electrode that binds to a target, where a reporter group is detectedby the electrode towards the end of a voltage pulse and uses goldparticles on the electrode and biotin immobilization. U.S. Pat. No.6,346,387 discloses another mediator approach, but with a membrane layerover the electrode through which a transition metal mediator can pass.U.S. Pat. No. 5,945,286 is based on electrochemistry with intercalatingmolecules. U.S. Pat. No. 6,197,508 discloses annealing single strands ofnucleic acid to form double strands using a negative voltage followed bya positive voltage. Similar patents include U.S. Pat. No. 5,814,450.U.S. Pat. No. 5,824,477, U.S. Pat. No. 5,607,832 and U.S. Pat. No.5,527,670 which disclose electrochemical denaturation of double strandedDNA. U.S. Pat. No. 5,952,172 and U.S. Pat. No. 6,277,576 disclose DNAdirectly labeled with a redox group.

Several patents address devising cartridge-based features or devices forperforming nucleic acid analyses, these include for example a denaturingdevice U.S. Pat. No. 6,485,915, an integrated fluid manipulationcartridge U.S. Pat. No. 6,440,725, a microfluidic system U.S. Pat. No.5,976,336 15 and a microchip for separation and amplification U.S. Pat.No. 6,589,742.

Based on the forgoing description there is a need for a convenient andportable analysis system capable of performing nucleic acid testing

OBJECTS OF THE INVENTION

An object of the invention is to provide an integrated nucleic acid testcartridge capable of performing extraction, amplification and detection.

A further object of the invention is to provide an integrated nucleicacid test cartridge with optical and electrochemical detection.

A further object of the invention is to provide an integrated nucleicacid test cartridge with an extraction step based on filter extractionor on particle transit through a layer that is immiscible with anaqueous fluid.

A further object of the invention is to provide an integrated nucleicacid test cartridge capable of performing extraction and amplification.

A further object of the invention is to provide an integrated nucleicacid test cartridge capable of performing amplification and detection.

An object of the invention is to provide an integrated cartridge fornucleic acid testing that operates in conjunction with a readerinstrument.

An object of the invention is to provide an integrated nucleic acidtesting system and method suitable for analyses performed at thebedside, in the physician's office and other locations remote from alaboratory environment where testing is traditionally performed.

An object of the invention is to provide a device and method of nucleicacid extraction from a sample with a purification step involvingparticle transit through a layer that is immiscible with an aqueousfluid.

An object of the invention is to provide a device and method offilter-based nucleic acid extraction from a sample with an elution stepprior to amplification.

An object of the invention is to provide a simple method and componentfor separating nucleic acid from a sample suitable for integration intoa device for performing genetic analyses.

An object of the invention is to provide electrophoretic separation ofprimers from amplicons after amplification capable of integration with anucleic acid testing cartridge.

An object of the invention is to provide a DNA polymerase enzyme thatgenerates the most synthesis in the shortest time period, therefore aDNA polymerase with an elongation rate of over 100 bases per second or aprocessivity rate of over 300 bases.

It is another object of the invention to provide a DNA polymerase enzymethat functions in a miniaturized thermocycler device in a short timeperiod.

SUMMARY OF THE INVENTION

In one embodiment, the invention is directed to a nucleic acidseparation method, comprising: exposing a sample comprising cellscontaining nucleic acid to an aqueous mixture comprising a lytic reagentand one or more beads capable of binding the nucleic acid released fromsaid cells to form a nucleic acid-bead complex, and passing the nucleicacid-bead complex through a substantially immiscible liquid layer toseparate the nucleic acid from the aqueous mixture.

Another embodiment of the present invention is directed to a method oftransferring nucleic acid, comprising: contacting nucleic acid at afirst location with one or more beads to form a nucleic acid-beadcomplex in a liquid, and transporting the nucleic acid-bead complex to asecond location separated from the first location by an intermediarylayer, wherein said intermediary layer is substantially immiscible withthe liquid.

In yet another embodiment, a nucleic acid separation method is provided,comprising: introducing a sample comprising biological cells through afirst layer to a second layer comprising magnetic beads, wherein saidfirst layer is substantially contiguous with said second layer,incubating the sample for sufficient time to permit lysis of the cellsand form a nucleic acid-bead complex in said second layer: and applyinga magnetic field in proximity to the complex sufficient to move saidcomplex from said second layer through said first layer, therebyeffectively filtering said complex.

In a further embodiment of the present invention, a method forextracting and amplifying nucleic acid is provided, comprising:introducing a sample comprising biological cells through a first layerto a second layer comprising magnetic beads, wherein said first layer issubstantially contiguous with said second layer; incubating the samplefor sufficient time to permit lysis of the cells and form a nucleicacid-bead complex in said second layer; applying a magnetic field inproximity to the complex sufficient to move said complex from saidsecond layer through said first layer, thereby substantially removingsaid complex from said second and first layers; and introducing saidcomplex to a vessel containing a polymerase chain reaction (PCR)cocktail, wherein at least a portion of the nucleic acid elutes off saidmagnetic beads during a first heating cycle of the PCR.

Another embodiment of the present invention is directed to a method forextracting nucleic acid from a biological sample, comprising: applying abiological sample comprising cells containing nucleic acid to abiochemically inert filter positioned between a first location and asecond location in a conduit; retaining the sample on the filter forsufficient time to permit extraction of nucleic acid onto the filter;applying a wash fluid through the conduit, whereby said wash fluidsubstantially removes from the filter matter capable of interfering witha nucleic acid amplification reaction, while substantially retaining theextracted nucleic acid on the filter: and applying an aqueous solutionto the filter at a temperature above about 75° C. to elute the extractednucleic acid from the filter.

The present invention particularly addresses expanding opportunities forpoint-of-care diagnostic testing, i.e. testing that is rapid,inexpensive and convenient using small volumes of accessible bodilyfluids such as, for example, blood or buccal cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows nucleic acid purification in a tube using a lytic bufferlayer, a wax layer and magnetic beads.

FIG. 2 shows a polyacrylamide gel of PCR products with and without beadsand with and without blood, and also purified DNA controls.

FIG. 3 shows a polyacrylamide gel of PCR products with beads and blood.

FIG. 4( a)-(d) show different perspectives of the filter holder.

FIG. 5 shows PCR amplification of a buccal swab sample isolated from afilter.

FIG. 6 shows a topological representation of the integrated single-usedevice and its interaction with the instrument.

FIG. 7( a) shows a schematic of the PCR amplification method. FIG. 7( b)shows a schematic of PCR amplification without a self-annealing primerand FIG. 7( c) shows a schematic of PCR amplification with aself-annealing primer.

FIG. 8( a) shows a typical chronoamperometry output for PCR plusconjugate and conjugate alone, and FIG. 8( b) shows a typicalchronoamperometry output for control plus conjugate and conjugate alone.

FIG. 9( a) shows chronoamperometry of different amplicon concentrationsand FIG. 9( b) shows a plot of the steady-state current signal versusamplicon number.

FIG. 10 shows a schematic for rolling circle amplification (RCA).

FIG. 11 shows a schematic for strand displacement amplification (SDA).

FIG. 12( a)-(b) show two perspectives of an electrophoresis componentfor integration into a single-use device for nucleic acid testing.

FIG. 13( a)-(g) show an electrophoretic separation using a component forintegration into a single-use device for nucleic acid testing.

FIG. 14 shows an electrophoretic separation of a primer and an ampliconusing a component (as shown in FIG. 13) for integration into asingle-use device for nucleic acid testing, confirmed by a secondelectrophoresis gel.

FIG. 15 shows an oligonucleotide primer lacking CLAM-like features.

FIG. 16( a) shows the CLAM1 primer and FIG. 16( b) shows the CLAM2primer.

FIG. 17( a) shows an optical detection-based single-use cartridge wherean optical sensor is integrated into the device and FIG. 17( b) shows anoptical single-use cartridge where the sensing region is a cuvettefeature permitting detection with a light source and detector integratedinto the instrument.

FIG. 18 shows an extraction device containing a filter region integratedinto a single use cartridge for nucleic acid testing.

FIG. 19 shows a two-part cartridge with a separate extraction componentthat can mate with the amplification and detection component.

FIG. 20 shows a two-part cartridge with a separate detection componentthat can mate with the extraction and amplification component.

FIG. 21( a) shows a cartridge and instrument separately and FIG. 21( b)shows the cartridge inserted into the instrument.

FIG. 22 shows examples of optical detection chemistries.

FIG. 23 shows an extraction and amplification component where a siliconchip provides one of the walls forming the extraction and amplificationchambers.

FIG. 24 shows a single-use device with electrophoretic separation ofunused primers after amplification.

FIG. 25( a)-(b) show a cleavage reaction creating a “trigger event” forfurther amplification and detection.

FIG. 26 shows a schematic of the PCR amplification method whichdifferentiates between mutant and wild-type SNP sequences.

FIG. 27 provides a table of possible signal outcomes from ahemochromatosis test.

FIG. 28( a)-(b) show two views of a buccal sample device for directapplication of a buccal sample to a PCR chamber. This extraction andamplification device attaches to the detection cartridge.

FIG. 29( a)-(b) show a comparison of signal which increases relative tothe amount of control oligonucleotide.

FIG. 30( a)-(b) show the ability of the cartridge to discriminatebetween wild-type and mutant SNP sequences of hemachromatosis.

FIG. 31 shows an autoradiograph of 32P radiolabelled syntheticoligonucleotides demonstrating that the ExoI enzyme is an active 3′->5′exonuclease, which has the ability to reduce the molecular weight downto about 6-7 nucleotides in length.

FIG. 32( a) shows PCR with phosphorothioate primers discriminatingbetween wt/mut DNA templates using a 10% non-denaturing polyacrylamidegel; 6 μL sample+1.6 uL LD→6 uL loaded in each well (45 min SYBR Goldstain, photo-negative, experiment HFE 84-2. T_(hyb) 68° C. The sevencolumns were loaded as follows: 1 wildtype-selective PCR primer presentwith wildtype DNA template, generates anticipated ^(˜)150 bp band; 2wildtype-selective PCR primer present with mutant DNA template, does notgenerate anticipated ^(˜)150 bp band: 3 a 10 base-pair ladder, prominentbands at 100, 330 and 1660 bases; 4 mutant-selective PCR primer presentwith wildtype DNA template, does not generate anticipated ^(˜)150 bpband: 5 mutant-selective PCR primer present with mutant DNA template,generates anticipated −150 bp band; 6 a 10 base-pair ladder, prominentbands at 100, 330 and 1660 25 bases; and 7 both selective PCR primerspresent with wildtype DNA template, does not generate any band. FIG. 32(b) shows the related chronoamperometry plot

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Nucleic AcidSeparation Methods and Apparatuses Based on Magnetic Particles

The present disclosure demonstrates a rapid and simple protocol forisolating genomic DNA from whole blood for the primary purpose ofperforming an amplification reaction, e.g. polymerase chain reaction(PCR). The present method has the advantage of exhibiting a significantreduction in the common inhibitors of PCR, e.g. hemoglobin, found inprior art rapid DNA extraction protocols. In blood samples, addedanticoagulation reagents such as chelating agents, heparin, EDTA andcitrate can also act as inhibitors. The present method eliminates theseinhibitors and other naturally occurring chelating agents as well asenzymes and proteins that can damage nucleic acid templates. It isimportant to note that this technique is also applicable to othersources of nucleic acid material, e.g. buccal swabs, urine, and othertissue samples, and can also be used in conjunction with otheramplification methods.

By contrast with the prior art, for example that found in DynabeadsGenomic DNA Blood kit (Prod. No. 634.02, Dynal Biotech Corp.), and alsoUS patent 2003/0180754A1 where nucleic acid extraction takes 30-40minutes, the present method reduces the time required for reproducibleDNA extraction to less than about 5 minutes and preferably and typicallyto about 2 minutes. This is a significant improvement when consideringgenetic analyses where the speed with which a result is obtained iscrucial, e.g. the identification of highly infectious agents. It is alsoapplicable to testing in the physician's office environment, or even atthe bedside, where it is desirable to obtain a sample from a patient anddeliver a result during a single physician visit.

The present method preferably uses coated beads, with an inner-core thatis a paramagnetic material and a lysing and binding buffer. When a lysedcell solution containing genomic DNA is mixed with beads of thepreferred embodiment, the surface chemistry on the beads weakly bindsDNA with low specificity due to a strong negative surface charge, thuscreating a bead-DNA complex. The preferred surface coating is acarboxylic acid coated surface and the paramagnetic beads typically havea 2.8 um diameter, though beads in the diameter range of about 0.1 to100 um can be employed. Alternative anionic coatings for the beadsinclude the following materials including very small diameter glassbeads (e.g. Glass Milk). Whatman phosphocellulose and DEAE resin (e.g.DE52).

While non-magnetic beads may be used, it is certainly advantageous touse magnetic beads as these beads may be drawn to the side of a reactionvessel and held against the side by means of a magnet. This can occurwithin a short period of time, provides a means for concentrating thebead in one location and provides a means for moving and manipulatingthe beads. The magnetic field may be provided by a permanent magnet orby electromagnetic means, as is well known in the art.

In an example that uses a standard polypropylene PCR tube, a standardlytic buffer (Dynal Biotech Corp.) containing: water 60-100% wt, sodiumchloride (NaCl) 10-30%, lithium chloride (LiCl) 5-10%, tris-HCl 1-5%,lithium dodecylsulfate (LiDS) 0.1-1%, EDTA 0-1%, and dithiothreitol(DTT) 0-0.1% was modified to include NaOH reagent at a final alkalineconcentration of 0.65M. Other lytic buffers known in the art may also beused with the appropriate addition of base, e.g. NaOH. Whole blood (10uL) was then added directly to the alkaline-modified lytic buffer withDynabeads (23 uL). This induced the lysis of blood cells in about 15seconds of manual pipette mixing, followed by about 15 seconds of dwelltime for the adsorption of genomic DNA onto the beads. The bead-DNAcomplex was then captured against the side of a tube with a permanentmagnet, which takes less than about 15 seconds. The entire supernatantof lysed cells was then removed by pipette. A wash buffer 50 uL). e.g.Dynal wash buffer (from a Dynal kit) was introduced by pipette and usedto rinse the bead-DNA pellet that was captured against the tube wall.The wash solution was then entirely removed by pipette while the pelletremained captured against the tube wall. The remaining bead-DNA pellet(1-2 uL equiv. volume) was then removed and added to a new tube with aPCR cocktail (^(˜)25 uL) comprising polymerase enzyme, primers, dNTPsand buffer along with a mineral oil overlay (^(˜)10 uL) and placed intoa conventional thermocycler. The total duration of this extractionprocess was found to be about two minutes. Note that it is demonstratedbelow that this novel purification protocol overcomes the problemassociated with inhibitors of a PCR reaction remaining in the extract.

In a preferred embodiment, the extraction method employs alkaline lyticbuffer, magnetic beads and also a wax or oil-filtering medium. Again,the method can be performed as a manual procedure, as described here, oras the basis of an automated analysis in a disposable device. The use ofwax or oil as a filtering medium overlaying the lysed-cell bead-DNAcomplex mixture eliminated the need for further fluid movement andassisted in purifying the bead-DNA complex. For instance, blood wascombined with the lytic buffer and beads and the resulting DNA-beadcomplex was pelleted and drawn through an upper filtering layer with apermanent magnet, thus selectively separating the complex from the bulkof solution. This is illustrated in detail in FIG. 1.

FIG. 1 shows a tube 1 contains a wax filtering medium 2 above a lyticbuffer 3 and magnetic beads 4. Typically the tube is stored at ambienttemperature, so the tube is first heated to melt the wax. Generally,this is a temperature change to above about 35° C. Blood 5 is introducedwith a pipette 6 and the blood is well mixed so that cells lyse in thebuffer. Nucleic acid 7 then binds to the beads via non-specific surfacebonds. A magnet 8 is then used to draw the beads and some extra lysedmaterial and buffer to side of the tube to form a pellet. The magnet isthen moved along side the tube to draw the pellet upwards through thewax layer. It has surprisingly been found that this effectively filtersthe pellet, as excess aqueous fluid is excluded by the greater surfacetension of the wax. Optionally, after this step, the wax may bere-hardened by removing the heat. The resulting bead-nucleic acid pelletremains trapped in a thin layer of wax easily accessible at the side ofthe tube, while the lytic buffer and blood remains trapped below thewax. The bead-nucleic acid pellet can then be removed from the side ofthe tube and introduced to a new tube with the PCR cocktail present. Thenucleic acid elutes off the bead during the first heating cycle of PCR,as it has been found that water at a temperature of above 800 C issufficient for elution. It has also been found that neither the beadsnor the wax interfere with PCR.

Ideal characteristics of waxes for this application include waxes whichmelt from a solid to a liquid at between 25 to 450 C. Further, thesepreferred waxes do not significantly evaporate at temperatures in therange 60 to 900 C. When these waxes are solid they prevent movement ofbead and other solutions that are trapped by their presence, however,when these waxes are in a liquid state their viscosity is sufficientlylow to permit passage of magnetic beads under a magnetic field. Thewaxes also have the property of being compatible with reagents for DNAamplification. Four examples of waxes that can be used in the presentinvention are heneicosane (98%, m.p. 40-42° C., Sigma), docosane (99%,m.p. 43-450 C, Sigma), tricosane (99%, m.p. 48-50° C., Sigma) andtricosaheneicosane. The preferred wax is heneicosane. Other organicliquids that can be used to form the barrier layer through which thebeads pass include silicone oil and mesitylene.

FIG. 2 demonstrates the successful removal of a purified DNA sample fromblood using the beads transiting through wax process, with the presenceof the anticipated bands (gel lanes 5 and 6 matching lane 2). Thisfigure shows a polyacrylamide gel of PCR products with and without beadsand with and without blood and also purified DNA controls. Note that theband labeled “*” represents the anticipated base-pair length forsymmetrical PCR with a modified wild-type Hemachromatosisoligonucleotide primer set prepared on a known wild-type alleles ACDblood tube sample. The positive control (lane 2) also represents genomicDNA purified using a Qiagen commercial kit for sample preparation(wild-type 15 alleles) and the negative control (lane 1) features withno DNA added to the PCR cocktail. In this example, PCR was performed ina conventional thermocycler, with a mineral oil overlay, using 30cycles. A volume of 10 uL of sample plus 2 uL of loading dye was addedinto each well of a 10% non-denaturing polyacrylamide gel, 1×TBE buffer,as shown in FIG. 2.

FIG. 3 contrasts the successful removal of purified DNA from blood usingthe beads transiting through wax protocol (gel lanes 1-4) to theprotocol without using the wax as a filter medium (gel lane 5). The bandlabeled “*” represents the anticipated base-pair length for symmetricalPCR with a modified wild-type Hemachromatosis oligonucleotide primer setprepared on a known wild-type alleles ACD blood tube sample. PCR wasperformed in a conventional thermocycler, with mineral oil overlay,using 30 cycles. A volume of 10 uL of sample plus 2 uL of loading dyewas added into each well of a 10% non-denaturing polyacrylamide gel,1×TBE buffer, as shown in FIG. 3.

The principles demonstrated by the above description can be incorporatedinto an individual nucleic acid extraction device based on manualmanipulations of the type shown in FIG. 1, or into an automatic deviceas described below, where the user only needs to add the sample to thedevice and all the other steps are performed automatically.

Nucleic Acid Separation Methods and Apparatuses Based on AbsorbentFilters

An alternative approach to quickly extract and isolate nucleic acidsfound in bodily fluids is provided. It is based on the use of filtermaterials. The disclosed devices and processes significantly improveupon the existing art by marrying chemically impregnated solid-substratetechnologies to a miniaturized filtering apparatus. It also convenientlyminimizes the time for extraction of an amplifiable quantity of genomicDNA from a low volume of bodily fluid. While the device may be used asan individual separation device, it is particularly amenable tointegration into a disposable cartridge device for DNA isolation,amplification and optionally detection.

The individual device can be used, for example, in clinical and researchenvironments as a rapid means for taking a small volume of fluid, suchas blood or buccal cells, and quickly isolating DNA amenable toamplification. Alternatively, when incorporated into a disposablecartridge, microfluidic elements are used to automatically move thesample within the cartridge and to affect the extraction process. Bothapplications are described.

The primary features of the device and method combine; (i) rapid nucleicacid isolation, typically in less than two minutes, (ii) elementsamenable to incorporation in a disposable cartridge, (iii) generation ofeither bound or unbound nucleic acid in a form compatible withamplification, (iv) utilization of small sample volumes, e.g. blood,buccal cells and tissue, and (v) utilization of small volumes of otherliquid reagents to perform the operation.

Regarding the device, the supporting structure of a low-volume filterholding apparatus was used for the placement of a chemically-impregnatedsolid-substrate matrix. It functions as a filtering layer that extractsand isolates DNA from an applied sample by retaining these nucleic acidswithin its matrix. The filtering matrix was impregnated with lytic saltsand optionally detergent, which after the binding step is then flushedor washed with a solvent, preferably distilled or sterile deionizedwater, to remove common inhibitors of amplification and to rinse awaydenatured proteins. The filter retaining nucleic acids from the samplecan then be removed from the supporting apparatus and directly appliedto amplifying reagents, e.g. PCR. This can be done using the wholefilter disc, or a portion thereof, depending upon the quantitativerequirements for DNA. Where desirable the nucleic acid material may beeluted from the filter preferably using deionized water at a temperaturein the range 75 to 950 C. Other eluting reagents include dilute neutralbuffers, such as 10 mM Tris at pH 7 and 5 mM to 20 mM sodium orpotassium phosphate buffers. Alternatively, a filtering matrix can beincorporated in a disposable nucleic acid testing cartridge, asdescribed below.

The preferred embodiment of the individual extraction device isdescribed as follows: The chemically-impregnated filter is a disccomposed of a reproducible thin matrix that is biochemically inert,preferably a commercially available filtering paper. The lytic salts andoptionally a detergent are dispensed onto the surface of the filter andthen dried within the matrix. As a practical matter, the size of thefilter-disc is restricted by the outer-diameter of the filter holder,and must be wider than the channel through which the wash fluid passes.Chemical impregnation is by means of a liquid cocktail containing achaotropic salt, with or without detergent, a weak basic buffer, and achelating agent. The cocktail is dispensed onto the filter-disc, driedand then the filter is stored in a sealed environment until use.

In the preferred embodiment, the filter holder device provides rigidsupport to the filter-disc (optionally with a placement-assistinggasket) with a central small-diameter channel through which the washfluid may pass from one side of the filter-disc to the other. The devicecontains both an inlet and an outlet on opposite sides of thefilter-disc to allow for the introduction and later removal of the washfluid. Its construction material should be biochemically inert,preferably a molded plastic. It is designed to be disposable, but itoptionally could be reusable if properly cleaned. e.g. autoclaved. Thefilter base-pad is a subcomponent that assists in the proper placementof the filter-disc in line with the wash fluid channel. Optionally afilter-positioning gasket may be employed for sizes of filter that aresmaller than the internal diameter of the device. For example a thinadhesive layer with a central hole that holds the filter-disc onto thefilter base-pad over the channel may be used. In this embodiment, adouble-sided adhesive tape with a central hole slightly smaller than theouter-diameter of the filter-disc is preferred. Wash fluid is preferablydistilled water and is used to remove chemical inhibitors ofamplification.

As is well known in the art, conditions of sterility and biochemicalinertness are intrinsic to the choice of materials employed for theconstruction of the device, the handling of fluids and the source of thewash fluid. Samples, e.g. bodily fluids, can be introduced to thefilter-disc through the inlet of the filter holder, or onto thefilter-disc before assembly into the device, provided care is taken toensure sterility.

In one embodiment, the filter holder can be a Swinnex filter holder,preferably the 13 mm diameter version (Millipore Corp.), which is alsoprovided with a Teflon™ gasket and is constructed of moldedpolypropylene. In a preferred embodiment, a modification was performedupon the filter holder where additional acrylic pieces are cut toexactly fit the void spaces inside both the top and bottom pieces of thefilter holder. These pieces are preferably held in place with adhesive,e.g. Loctite epoxy glue, and have a drilled central channel of a smallerdiameter than the standard device. The inlet to the filter holder canalso optionally be modified with an end piece from an Eppendorf 100 μLpipette tip that is held into position with adhesive.

The filter positioning gasket is preferably a double-sided adhesive tapegasket (iSTAT Canada Ltd.), laser cut to a thickness of about 25 um on aPET film base with about 75 um of a rubber-acrylic hybrid adhesive,sandwiched between two polyester liners for protection. A two-sidedadhesive has the advantage of providing a better seal of the filterholder during the washing procedure. Note that the polyester liners areremoved during assembly of the device to expose the adhesive.

The filter disc is preferably Whatman 4 Qualitative Grade plaincellulose paper, (Whatman Inc.), with the following manufacturer'sspecifications; particle retention greater than 20-25 μm, coarseporosity, filtration speed ASTM 12 sec. Herzberg 37 sec., and a smoothsurface. Other similar filter materials and grades may be used includeWhatman 3 MM, Pall GF A/B. Texwipe (cleaning cloth). Whatman 1. Whatman3, Whatman 4, Whatman 6 and Pall 1660 membranes.

Chemical impregnation of the filter is preferably with a liquid cocktailthat contains chaotropic salts, preferably a guanidinium salt such asguanidine isothiocyanate, with or without detergent preferablyTriton-X100™, a weak basic buffer preferably TRIS, and a chelating agentpreferably EDTA. Alternative reagents include guanidinium salts (e.g.guanidinium hydrochloride and guanidinium thiocyanate), non-ionicdetergents and chelating materials. The cocktail is applied to Whatman 4paper in solution for minimal loading of approximately 3.75 μL/cm² of 2Mguanidine isothiocyanate, 1% Triton XI00, 10 mM TRIS buffered to pH 8.8and 2 mM EDTA. The cocktail is then dried under a heat lamp (Philips.Heat-Ray 250 w infrared) about 5 cm below the light surface for 3minutes, then cooled at room temperature for a minimum of 10 minutes andstored in a sterile centrifuge tube until use. Note that where theintended sample material is blood, it has been found that impregnationwith a solution of 200 mM NaOH can be substituted for all the reagentsused in the cocktail solution. Other strong basic solutions can also beused e.g. KOH.

By way of demonstration, two different bodily fluids have been used forthe extraction of genomic DNA. These are (i) white blood cells within awhole blood sample, that are untreated by either chelating oranticoagulation agents, and (ii) buccal cells obtained from a cheekswab. When utilized as described below, the present device can extractamplifiable DNA from both fluids with a minor variation in the protocol.Based on this disclosure, those skilled in the art will recognize thatother types of sample containing nucleic acid may also be extracted bymaking further minor variations in the protocol.

The component elements of filter holder are shown in FIG. 4( a) in sideview, FIG. 4( b) exploded side view, FIG. 4( c) top view and FIG. 4( d)with a void volume insert. The device comprises a filter holder top 20and bottom 21, an inlet channel 22, void spaces 23 and 24, a filter disc25 on a filter disc base and an outlet channel 26. In the preferredembodiment, as shown in FIG. 4( d), a lower volume modification employsa void-filling structures (27, 29) and an inlet adaptation element 28 tofacilitate better transfer of fluid into the narrower central channelvia inlet 22. The lower volume device requires the filter-disc to bepositioned with a filter gasket attached to adaptation element 29. As apractical matter, the device is prepared in a sterile workingenvironment and tools to prevent cross-contamination of nucleic acidsand enzymes are used.

When using a 13 mm filter-disc 25, about 3-10 μL of bodily fluid can beapplied to the chemically-impregnated filter surface, whereas thelower-volume modified device, with a 4.8 mm filter disc functions wellwith 1-3 μL of fluid. Sample application can be achieved with theassembled device through the inlet port, or directly onto the filterprior to assembly. Where a buccal swab is acquired with a cotton swab,it can be wiped onto the filter disc or washed onto the filter discthrough the inlet port. It has been found that another method forisolating buccal cells is by using a commercial mouthwash, e.g. Scopebrand. A few microliters of used mouthwash can then be applied into thedevice.

Regarding removal of interferents, it was found that sterile water atambient temperature performs satisfactorily as a wash fluid as it iscapable of flushing interferents through the filter-disc withoutremoving nucleic acids from within the matrix of the disc. When water ispumped from a dispensing tip positioned for a tight seal at the inlet tothe filter holder, it flushes through the filter-disc washing the sampleand passing through to the outlet. For buccal cell samples, a singleflush of 20 μL of sterile water per μL of sample is sufficient. Forblood samples, 20 μL of sterile water per μL sample is preferablyflushed through the filter and repeated three times. Alternatively asingle volume passed forward and backwards thrice is sufficient. As analternative to sterile water the following sterile buffer solutions maybe used. 10 mM Tris at pH 7 and 5 mM to 20 mM sodium or potassiumphosphate.

After the washing procedure the filter-disc retains an amplifiablequantity of DNA. It can then be removed from the filter holder andemployed in an amplification reaction. It has been found that a 4.76 mmdiameter disc can be employed in a 100 μL PCR amplification directly,whereas a 13 mm disc is optimally cut into smaller portions. In analternative embodiment the nucleic acid material can be eluted from thefilter by using hot deionized water or various buffer solutions and thenintroduced into an amplification device. In another embodiment thefilter process is integrated into a disposable device for nucleic acidtesting, as described below. FIG. 5 demonstrates the effectiveness ofthe method and filter holder device, showing PCR amplification of abuccal swab sample. After the extraction process, the filter was removedfrom the device and placed into a 100 uL PCR reaction chamber using twoprimers specific for the hemachromatosis gene (Hfe). Once theamplification process was completed, material was applied to lane 1 of a10% acrylamide 1×TBE electrophoresis gel. As expected this generated a390 bp (base pair) fragment indicated by the arrow. Note that controllane 2 contained a 100 bp ladder and lane 3 contained water as anegative control.

It is understood that the manual procedure described above can form thebasis for the design of an extraction module included and integratedwithin a disposable device for performing genetic analyses, or be aseparate module that delivers an extract to a disposable device.Delivery can be for example by pipette transfer or by mating features500, 520 and 521 on each that facilitate transfer (see FIGS. 19 and 20).Such devices are described in detail in the section addressing anintegrated single-use device for nucleic acid testing.

Detailed Description of Amplification Methods

In the present invention, where electrochemical detection is preferred,the main objective of the nucleic acid amplification step is to generateabout a 0.01 picomolar concentration of detectable nucleic acid from thetarget molecule, as it has been found that this is in the range of thelower detection limit of a sandwich assay with enzymatic amplificationand electrochemical detection. The desired one picomolar concentrationof fragment is based on Avogadro's number (1 mole=6×10(23) molecules),where 1 pmol equals 6×10(23)×10(−12), or about 10(12) molecules. If, asis known, one microliter of blood contains about 5×10(3) molecules ofDNA, then one milliliter, which is a reasonably accessible samplevolume, contains 5×10(6) molecules, or roughly about 10(7) molecules. Togo from the amount of DNA in 1 ml of blood to 0.01 pmol of DNA requiresan amplification of about 10(3) fold. This is certainly achievable usingseveral well-known amplification techniques. Performing a similarcalculation, for a different sample types and sample volumes, todetermine the degree of amplification will be apparent to those skilledin the art.

In alternative embodiments of a single-use cartridge where opticaldetection is used, again the objective of the nucleic acid amplificationstep is to generate a given molar concentration of detectable nucleicacid from the target molecule so as to be in the range of the lowerdetection limit of the given optical methods. Such calculations will befamiliar to those skilled in the art. It is well known in the art thatthe ability to determine the concentration of a sample via opticaldetection is dependent on the background level of noise, the extinctioncoefficient of the optical compound to be detected, the optical system'selectronic gain, the volume of the sample and other parameters. A simplerelationship between the compound concentration and the absorbance ofthe sample can be expressed using the Beer-Lambert law (A=εcl), where Ais the absorbence, ε is the extinction coefficient, c is the molarconcentration of the sample, and 1 is the path length of the sample.Typically the length is 1 cm by definition, (though in the devicesdescribed below about 0.02 to about 0.4 cm is more typical). This makesthe absorbence related to the concentration using the constant of theextinction coefficient and usually permits detection limits within thepM range.

Polymerase Chain Reaction Amplification

The polymerase chain reaction (PCR) is well known for its ability tospecifically amplify regions of target DNA based on the primer sequenceschosen for the PCR reaction. A difficulty with processing this materialis in trying to detect the signal based on hybridization homogeneously.By definition, the PCR reaction generates blunt ended double strandedproducts. However, certain thermostable DNA polymerases possess polyApolymerase activity, which can be used to add an additional Anucleotide. While this has been used commercially for cloning purposes,the single nucleotide overhang is inefficient for hybridization. Asanother approach to attempt to use the PCR reaction for hybridization,recognition sequences for restriction endonuclease enzymes have beendesigned into the PCR primers. However, this is limiting, because itrequires additional enzymes which typically only generate shortoverhangs. As with mostly double stranded species, the PCR product isnot amenable to hybridization in homogenous reactions. To overcome thislimitation, a strategy which uses a limiting amount of one primer overthe other has been devised. An alternative is to have promoter regionsfor bacteriophage RNA polymerases (e.g. SP6). Limiting one of theprimers has drawbacks in that the efficiency of the amplification isreduced. Generating RNA with bacteriophage RNA polymerases requiresadditional reagents and generates labile RNA species for detection.

Here we describe a novel method of performing a PCR reaction bycombining DNA polymerase, a target nucleic acid and amounts of twomodified primers where the first modified primer has a sequence of basesto a region of the target. A polymerase blocking region is attached tothis primer which is linked to a single stranded hybridization region.The second modified primer has a sequence of bases to a second region ofthe target and also a polymerase blocking region and a second singlestranded hybridization region. A detectable moiety (e.g. biotin,fluorocein) is attached to one or both of the two modified primers. Torun the PCR reaction the mixture is cycled to generate multiple copiesof an amplicon incorporating the modified primers. In a second stepexcess unincorporated modified primers, with the detectable moiety, aresubstantially eliminated from the mixture. Several different methods areavailable and these are described below. The mixture is then added to acapture oligonucleotide which is complimentary to one or both of thesingle stranded hybridization regions to permit hybridization with theamplicon. In the last step the moiety associated with this hybridizationis detected directly, for example by optical detection of fluorocein.Alternatively, the moiety, e.g. biotin is exposed to and binds with astreptavidin-labeled enzyme, e.g. alkaline phosphatase and the enzymeactivity is determined either optically or electrochemically. Againseveral specific methods are possible and examples of these aredescribed below.

The reaction sequence is shown in FIG. 7( a), where 31 is the detectionmoiety, e.g. biotin, FAM, DNP, cholesterol, fluorocein, 32 is the firstsingle stranded hybridization region, 33 is the polymerase blockingregion, e.g. hexaPEG, 34 is the first PCR primer, 35 is the second PCRprimer, 36 is the second single stranded hybridization region, 37 is asecond detectable moiety, 38 is the double stranded nucleic acid targetsequence, 39 is a solid substrate, e.g. bead or surface, and 40 is ahybridization region complementary to 36.

The PCR primers, 34 and 35 are preferably synthesized using standardphosphoramidite chemistry and can include any nucleotide or modifiedbase which is amenable to DNA polymerase, except in the polymeraseblocking region 33. An example of a polymerase blocking region sequencecan consist of the spacer phosphoramidite18-O-dimethoxyltritylhexaethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (hereinafterreferred to as “HPEG”). This phosphoramidite generates ahexaethyleneglycol spacer region. Other spacer molecules with similarproperties can also be used for this purpose. Alternatives tophosphoramidite chemistry can be used including creating a 3′-3′ or5′-5′ phosphodiester backbone, as well as modified nucleotides asdescribed by Newton et al. (Nucleic acids research 21, 1155-62, 1993)and also U.S. Pat. No. 5,525,494.

Allowing PCR to proceed using these synthetic oligonucleotide primers inthe presence of the appropriate target and DNA polymerase withassociated components, generates a newly synthesized DNA molecule withincorporated single stranded regions 32 and 36. It has been found thatwhile the Taq DNA polymerase may be used, the preferred embodiment usesT. kodakiensis DNA polymerase which exhibits a significantly higherturnover number. This molecule can then be hybridized by means of 36 toa target sequence 40 on a solid support 39. The binding moiety regioncan then be used for generating a signal. For example by using biotin asthe binding moiety and using streptavidin conjugated to a detectionenzyme, e.g. horseradish peroxidase (HRP) and alkaline phosphatase(ALP).

The PCR primer also preferably contains a terminal phosphorothioatebond, preventing the exonuclease activity of T. kodakiensis KOD1 DNApolymerase from not discriminating allelelic differences in primers usedin SNP analysis based on the terminal base being different.

In the preferred embodiment using human genomic DNA isolated using thefilter holder device described above, two synthetic oligonucleotides(primers 1 and 2) were used to generate a region of the humanhemochromatosis gene (hfe) of approximately 390 bp in size. These wereoligo 1: 5′-ACTTCATACACAACTCCCGCGTTGCATAACT-HPEG-TGGCAAGGGTAAACAGATCC-3′and oligo 2:5′-56-FAM-AACAATACCACCGTAGCGATCA-HPEG-AACAATACCACCGTAGCGATCA-3′, where56-FAM is a fluorescent species and HPEG is a hexa PEG sequenceincorporated using an 18-0-dimethoxyltritylhexaethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite. In the oligo 1sequence, the sequence located 5′ to the HPEG spacer,5′-ACTITCATACACAACTCCCGCGTTGCATAACT-3′ is designated as SEQUENCE ID NO.1 and the sequence located 3′ to the HPEG spacer,5′-TGGCAAGGGTAAACAGATCC-3′ is designated as SEQUENCE ID NO. 2. In theoligo 2 sequence, the sequence located 5′ to the HPEG spacer,5′-AACAATACCACCGTAGCGATCA-3′ is designated as SEQUENCE NO. 3 and thesequence located 3′ to the HPEG spacer, 5′-AACAATACCACCGTAGCGATCA-3′ isdesignated as SEQUENCE ID NO. 4.

To demonstrate the use of these primers, a buccal cell DNA sampleoriginating from mouthwash (Scope brand) was used. A volume of 3 μL ofthis bodily fluid was dispensed onto a 5 mm diameter disc punched fromWhatman 4 filter-paper impregnated with 3 μL of lytic salt and detergentsolution comprising 2M guanidinium isothiocyanate, 1% Triton-X-100, 10mM Tris buffer at pH 8.8 and 2 mM EDTA. After extraction, thefilter-disc was placed immediately into a 0.5 mL MβP Easystart PCRreaction tube (Fisher Scientific. PN 21-402-49) designed to be filled to100 μL. The tube is supplied with 50 μL of fluid under a waxlayer togive a final concentration of the following reagents in 100 μL ofaqueous solution; 2 mM MgCl₂, 20 mM Tris pH 8.4, 50 mM KCI and 0.2 mMdNTP. A 47 μL upper-layer reaction mixture was added to give a finalreaction concentration of primers 1 and 2 of 0.31 pM, described(Integrated DNA Technologies Inc). This aqueous solution also contained5U Vent (exo-) polymerase (New England Biolabs) and 0.1% Triton-XI-00.The amplification reaction was performed in a Techne TechgeneThermocycler. The sequence was amplified using 3 cycles of 97° C. for 3min, 60° C. for 1 min and 72° C. for 1 min, followed by 36 cycles of 97°C. for 1 min and 62° C. for 45 s. Samples resulting from theamplification procedure were then tested in single-use cartridges using100 μL aliquots. A complete description of the design elements ofdetection cartridge containing an electrochemical sensor is found injointly owned US 20030170881 incorporated here by reference. A generaldescription of chronoamperometry and other electrochemical methodsapplicable to sensors incorporated into single-use test cartridges isfound in jointly owned U.S. Pat. No. 5,112,455 incorporated here byreference.

The 100 μL aqueous aliquots were prepared as follows; 14 μL 1M NaCl. 1μL FITC-ALP conjugate 1/100 dilution, and 10 μL amplified DNA. TheFITC-ALP conjugate is a final concentration of 350 ug/ml. Alternativelya control oligonucleotide sequence was used in place of the amplifiedDNA. The control oligonucleotide sequence was manufactured as a positivecontrol for chronoamperometric detection. This single-stranded sequenceis analogous to 36 as shown in FIG. 7( a) and is complementary to region40 and contains a 36-FAM fluorescent species. Note that FIG. 7( b) showsthe undesired competition of a standard primer in the detection step,whereas with the clam-like primer, as in FIG. 7( c) this is obviated.The results from both of these samples are shown in FIGS. 8( a) and8(b). FIG. 8( a) shows the chronoamperometric reading for anti-FITC ALPconjugate alone versus the conjugate with amplicon hybridized to thesensor. FIG. 8( b) shows the chronoamperometric reading for anti-FITCALP conjugate alone versus the conjugate with a positive controloligonucleotide sequence.

The detection cartridge operated as follows, a 20 μL portion of the 100uL aliquots was loaded into an enzyme-linked DNA hybrid sensorcartridge, as described in jointly owned US 20030170881 and placed intoan i-STAT model 300 electrochemical analyzer (i-STAT Corporation). Thesensor cartridge contained multiple (2 or 4) amperometric sensors coatedwith specific DNA oligomers. In this example, the oligomers were5′-biotinylated oligonucleotides and were bound to streptavidin-coatedbeads which were adsorbed onto the sensor surface. One of the sensorswas coated with the complementary single-stranded DNA oligomer to one ofthe single-stranded portions of the PCR primers, as a control. Alsopresent within this cartridge was a separate anti-FAM-alkalinephosphatase conjugate.

In the preferred embodiment, the PCR amplified product and anti-FAM ALPconjugate dissolved into a single solution were brought into contactwith the DNA capture sensors. Note that alternatively the PCR productmay be contacted with the sensor first, followed by the conjugate. Inthe preferred embodiment, the double-stranded PCR products, containingboth single-stranded hybridization regions, binds to the capture regionon the amperometric sensor. Binding of the alkaline phosphatase labelcan occur either in solution before capture of the PCR product or afterit has bound to the bead. After a controlled period of time, typically 5to 15 minutes and at a controlled temperature preferably 37° C., thesolution is moved out of the sensor region and delivered to a wastechamber within the cartridge. A wash solution, containing substrate forALP, is brought over the sensor washing excess a FAM ALP conjugate awayfrom the sensor region. A trailing portion of the wash solution remainson the sensor and provides an electrogenic substrate for the ALP label.Note that in an alternative embodiment a wash solution may be usedfirst, followed by a second solution containing the substrate. Note alsothat where an optical sensor or other type of sensor is used, otherappropriate substrates are used. In the preferred embodiment, themeasured current at the capture sensor is essentially directlyproportional to the number of ALP labels present on the sensor. Anadjacent amperometric sensor which is not coated with the complementaryDNA binding sequence can be used as a control sensor to offset anynonspecific binding of the ALP reagent on the sensors, thus improvingthe detection limit. Alternatively a capture oligonucleotide with asequence different from the complimentary DNA binding sequence can beused as a negative control.

Referring to FIG. 8( a) and FIG. 8( b), these show the measured currentprofiles, or chronoamperometric output, from DNA cartridges. PCR productwith conjugate shows an increase in measured current, over conjugatealone, in FIG. 8( a). Here, competing unbound primers may be reducingsignal. A similar increase in signal is observed with thepositive-control oligonucleotide sequence that is labeled with 36-FAMspecies, as shown in FIG. 8( b). It has also been found that the netcurrent is proportional to the number of PCR amplicons in the sample,see FIG. 9( a), where the steady-state current is shown to increaseswith increasing amplicon concentration. These data are plotted in FIG.9( b).

The software used for the instrument 200 and 650 (see FIGS. 6 and 21) inthis example is a modified i-STAT 300 analyzer (i-STAT Corporation)which performs a series of steps in the detection process. In the firststep, the instrument makes contact with and identifies the cartridge,and then conducts a battery check and other internal instrument checks.It then initiates and completes a thermal cycle to heat the sensor chipto 370 C. The liquid containing the amplified target is thenpneumatically pushed from conduit 125 into the sensor chamber 126 topermit the capture steps. A push pin 213 in the instrument then makescontact with element 135 during the second motor motion of theinstrument causing the analysis fluid 134 to be dispensed from theanalysis pack into the analysis into conduit 125 which acts a temporaryholding chamber. The temperature set-point for the sensor chip is thenincreased to 47° C. and a conductivity sensor on the chip isinitialized. The target liquid is then pushed back and forth over top ofthe capture oligonucleotide beads to effect efficient capture of theamplicon. This step takes about 3 to 9 minutes. Note that theconductivity sensor is used to monitor the position of the fluid duringthis capture process. Before the last two oscillations, the software inthe instrument causes the heating of the chip to be turned off and theremaining cycles are conducted at ambient temperature. The liquidcontaining the uncapture amplicon is then moved slowly to the sampleinlet side of the waste chamber 137, and the sensors are set to collectdata at a poise potential of +30 mV vs. Ag/AgCl electrode (at 2 pA/bit).As this liquid is pushed into the waste chamber a locking wick mechanismcloses a vent when it becomes saturated. This mechanism is of the typedescribed in jointly owned US 20030170881 which is incorporated here byreference. The software then causes the instrument to actuate thecartridge such that analysis fluid is drawn across the sensors to washthe remaining unbound material from the capture oligonucleotide, leavinga thin layer of analysis fluid containing p-aminophenol phosphate whichcan react with the enzyme and be oxidized at the electrodes. Currentgenerated as a function of time is recorded, as shown in FIG. 9( a), andcan be used by the software algorithm to display a result.

It is known in the art that ExoI can be used to degrade un-incorporatedsingle stranded oligonucleotides in DNA sequencing reactions, however itwas not known if unnatural DNA, like the hexa-PEG region would bedegraded by the ExoI enzyme. To demonstrate that ExoI works on thisunnatural base, the experiment shown in FIG. 31 was performed. Thisfigure shows an autoradiograph of 32P radiolabelled syntheticoligonucleotides after ExoI treatment. In FIG. 31, the is 015oligonucleotide in lane 1 is the same as oligo 1 above. Theoligonucleotides labeled is 026 and is 027, like is 015 contained anHPEG spacer, while the is 020 oligonucleotide did not contain an HPEGspacer. FIG. 31 demonstrates that the ExoI enzyme is an active 3′->5′exonuclease, which has the ability to reduce the molecular weight downto about 6-7 nucleotides in length. Further, it can process past thehexa-PEG region and it is inhibited in the double stranded region of theclam primers. Therefore, it demonstrates that ExoI is not prevented frombeing an exonuclease with the hexa-PEG region.

In another embodiment of the invention, gene copy mutations, e.g.ZNF217, are detected by using both the target gene and one or morehousekeeping genes, e.g. actin or glyceraldehyde-3-phosphatedehydrogenase. This is accomplished with two sensors in the detectionchamber 126, with one for the target and the other for the housekeeper.Here, PCR primers are used to amplify both the housekeeping gene, aswell as the gene of interest. If ZNF217 is present in the same copynumber as the housekeeping gene, the level of signals is similar.However, when the ZNF217 gene is present in multiple copies, the levelof signal at the ZNF217 sensor is greater than at the housekeeping genesensor.

Another embodiment of the invention addresses genetic mutations whichcauses disease states includes gene expression mutations. Wildenhain etal., (1990, Oncogene, vol 5(6):879), describe the over-expression of theneu protein-tyrosine kinase, p185neu which is related to breast cancer.The c-Myc oncogene has been identified in many forms of cancer (Waikelet al., 1999, Oncogene, vol 18(34):4870). Other examples of oncogeneoverexpression were described by Ren (2004, Curr. Opin. Hematoi. Vol11(1):25). Over-expression mutations typically generate increased levelsof mRNA, thus to detect mRNA in this invention, an initial step of cDNAsynthesis is used prior to the PCR amplification. The synthesis of eDNAusing reverse transcription is well known in the art, includingamplification of this material by PCR. Using the PCR amplificationpreviously described, the presence of a quantity of mRNA present in acell can be determined by measuring the level of the signal. Comparingthe signal for a particular oncogene, for example Her2/neu to 5 ahousekeeping gene allows the discrimination of oncogene expression atnormal levels, or at levels indicative of a disease state, and inparticular with breast cancer in the case of Her2/neu.

FIG. 26 shows an alternative assay method schematic and experimentaldata for this method are shown in FIG. 32( a) by gel electrophoresis andFIG. 32( b) by chronoamperometry. Target nucleic acid (DNA or cDNA) 329is shown flanked by two regions where PCR and/or Clam primers bind 330and 333, with an intervening sequence marked by 331. During the PCRreaction, three primer sequences 31, 341 and 37 are added to thereaction mixture, wherein 31 and 341 differ by a single nucleotide attheir 3′ end 340 as indicated by 337 and 338.

Elements 31, 341 and 37 act as PCR primers, wherein region 336 forprimers 31 and 341 hybridize to region 330 on target molecule 329. Andregion 35 of molecule 37 hybridizes to region 333 on target molecule329. Primer 37 can function as a complementary PCR primer for primers31, 341 or both 31 and 341. Primer 37 also has the feature of a specificsequence of bases at region 35 wherein it hybridizes to target molecule329 at location 333. It has a DNA polymerase blocking group at 33,another unique region at 36 which will form a single stranded region forlater hybridization during detection and an optional binding moiety at37.

Clam primers 31 and 341 have many similar features, but also have somespecific differences. Both clam primers 31 and 341 have an optionaldetectable moiety at 334. This is for example a biotin molecule on 31and a FAM tag on 341. However, these are different for 31 and 341 toallow later discrimination of the molecule. Both clam primers 31 and 341have different designed single stranded binding regions 32 and 339respectively. In addition, both clam primers 31 and 341 have DNApolymerase blocking groups 33 and both clam primers 31 and 341 have apoint mutation designed into the fourth nucleotide base to assist in thediscrimination of single nucleotide polymorphisms, as described by Leeet al., (2004, Nucleic. Acids Research, vol 32(2):681). Newton et al.,(1989, Nucleic Acids Research, vol 17(7):2503), and European Patentapplication No. 89302331.7. As already mentioned, region 336 of bothclam primers 31 and 341 bind to region 330 on target molecule 329,wherein a single nucleotide mutation at 337 or 338 discriminates betweena single nucleotide difference. Both clam primers have a modifiedterminal phosphodiester bond at 340 that is resistant to 3′ to 5′exonucleases present in certain thermostable DNA polymerases, whichfurther assists in the discrimination of the two different molecules.This modified terminal phosphodiester bond can be a phosphorothioate orpeptide nucleic acid (PNA). The Clam primers also have the feature ofhaving intramolecular structure, which prevents the unincorporatedsingle stranded primer molecules from binding to a captureoligonucleotide 40 or 30 at the detection stage, but permits them tohybridize, to the capture oligonucleotides 40 and 30 if incorporatedinto a newly synthesized PCR amplicons.

In the first round of PCR after denaturation of the double strandedtarget to single strands, primer 37 and either clam primers 31 or 341 orboth 31 and 341 bind to target molecule 329. When only either clamprimer 31 or 341 binds to the target molecule 329 as is the case for ahomozygote, the single nucleotide on both copies of two chromosomes arethe same. When both 31 and 341 bind to two separate molecules of target329 as is the case for a heterozygote, one chromosome has one singlenucleotide base sequence, whereas the other chromosome has a differentsingle nucleotide base sequence as is found in single nucleotidepolymorphisms. This incorporates clam primers 31 or 341 or both 31 and341, as well as the PCR primer at the other end, 37 and the newlysynthesized intervening region 331.

PCR amplification is allowed to proceed for between 15 and 50 cycles togenerate newly synthesized amplified molecules. In FIG. 26 we show anamplicon 344 with Clam primer 341 incorporated. This is done forillustrative purposes. If the other mutation were present, or if therewas a different sequence on either of the chromosomes, an amplicon with31 incorporated would be found. For simplicity, only the amplicon with341 is shown in the figure.

During the detection step of the process, the newly synthesized PCRamplicon 344 with Clam primer 341 and PCR primer 37 incorporated bindsto capture oligonucleotide 40 at region 339 based on the nature ofcomplementary sequences binding to each other. Sequence 339 does notbind to a different physically separated capture oligonucleotide 30which possesses a different sequence. Both capture oligonucleotides 30and 40 are bound to a solid .substrate or beads as indicated in 39.

The detection of this hybridized complex can either be detected by aconjugate molecule which binds to binding moiety 334 in molecule 37, oranother single stranded oligonucleotide 318 binding at region 343 withregion 36 on molecule 344 having its own detectable moiety 342 which canbe detected by a conjugate molecule. The conjugate molecule has twofeatures: (i) a region that binds to the binding moiety 334 or 342, and(ii) a detection region. An example is an antibody specific for the FAMbinding moiety, which has been modified with an alkaline phosphataseenzyme as the detection element.

Alternative Amplification Methods

An alternative embodiment of this method using the same detectioncartridge can be used to perform a non-PCR nucleic acid amplificationassay. A schematic for rolling circle amplification (RCA) is shown inFIG. 10 and one for strand displacement amplification (SDA) in FIG. 11.Not that the component elements correspond to those described for PCR asshown in FIG. 7( a). Both assays require a short ssDNA fragment with a3′-OH moiety (308 and 310) made from the target, as shown by means oftwo different methods in FIG. 25. FIG. 25( a) shows a triggering eventmethod, e.g. SNPase and cycling probe, and FIG. 25( b) 25 shows theInvader™ method.

The same reagents are used as in the above section, however only onemodified primer comprising a sequence of bases to a first region of saidtarget nucleic acid is required. Again the mixture is cycled to providemultiple copies of an amplicon incorporating the 30 modified primer,followed by substantial elimination of any excess unincorporatedmodified primer from the mixture. Several methods can be used asdiscussed below. The mixture is then exposed to a captureoligonucleotide complimentary to the single stranded hybridizationregion, followed by hybridization of the single stranded hybridizationregion of said amplicon incorporating said modified primer, with thecapture oligonucleotide. Again the final step is detecting said moietyassociated with said hybridization, e.g. electrochemical detection of anelectroactive species generated by alkaline phosphatase. In thepreferred embodiment, primers are attached to the polymerase-blockingregion which, in turn is attached to a single stranded hybridizationregion.

For the rolling circle amplification strategy, the 3′-end of the primerhas a blocking region, which could include a phosphate or a dideoxynucleotide. A cleavage reaction similar to that found for the cyclingprobe reaction or the SNPase assay occurs, removing the blocking moiety,as shown in FIG. 25( a), comprising target DNA 300 and reagents 301, 302and 309 participating in reaction 306. Pre-made circular molecules canbe added to the reaction mixture. Extensions cannot occur with blockedprimers, but do occur to cleaved primer molecules. The cleaved primergenerate long single stranded molecules with duplications of specificregions complementary to the pre-made circular molecules. Syntheticoligonucleotides with detectable moieties are included in the mix,wherein the oligonucleotides are complementary to a region of the singlestranded DNA, which can be found multiple times along the singlestranded DNA. One region of the primer, which is single stranded andunique, binds to a capture oligonucleotide region. As this region is notcomplementary to the pre-made circular DNA, there is no competition ofthis region with the capture oligonucleotides. As shown in FIG. 10, inthe rolling circle assay the ssDNA 3′-OH moiety (308, 310) binds to therolling circle reagent (311, 315) via reaction 312. Cycling incorporatesa string of moieties 316 attached starting at the 3′ end of 308 or 310,to produced 314. Detection of element 314 is achieved by binding its5′-3′-OH region to complementary element 40 immobilized on bead 39 andlabeled polynucleotide 317 complementary to 316. The label is thenrecognized by an antibody bound to alkaline phosphatase 318.

An alternative embodiment of this method using the same detectioncartridge can be used to perform a non-PCR nucleic acid amplificationassay. A schematic for strand displacement amplification is shown inFIG. 11. Note that component elements correspond to those in the PCR asused as in FIG. 7( a). Similar reagents are used as those describedabove, however the SDA primer must first be provided in anon-amplifiable format, which is converted to an amplifiable format. Oneapproach to accomplishing this is to provide a primer with a blocked3′-end block, for example using a 3′-terminal dideoxy sequence. Atrigger event then occurs, which cleaves off the blocking 3′-end. Oneexample of a trigger event could be an Invader reaction (Kwiatkowski RW, Lyamichev V, de Arruda M, Neri B. Clinical, genetic, andpharmacogenetic applications of the Invader assay. Mol Diagn. 1999;4:353-364.), where the flappase activity cleaves at the hybridizedjunction of the blocked primer with the presence of genomic targetnucleic acid, providing an available 3′-10 hydroxy group. This is shownin FIG. 25( b) with target DNA 300 and reagent comprising 304, 303, 305and 309 participating in reaction 307. Alternatively, another example ofa trigger event is a cycling probe reaction (Duck et al., 1990,BioTechniques, vol 9(2): 142), where the presence of the genomic targetnucleic acid causes the cycling probe oligonucleotide to be cleaved at afour ribonucleotide sequence on the cycling probe oligonucleotide, inturn generating a free 3′-hydroxyl group. Another similar example is amismatch to the genomic target nucleic acid and a repair enzyme, whichas described for SNPase, generating a free 3′-hydroxyl group.

After the trigger event, which has generated a free 3′-hydroxyl group inthe primer sequence, a complementary strand displacement primer ispresent. This SD primer is complementary at its 3′ end for the primerdescribed above, which generated a 3′-hydroxyl group. In addition, theSD primer has 3′ to the 3′hydroxyl group complementary oligonucleotide aregion that when newly synthesized is cleaved by a Nickase restrictionendonuclease, as described in U.S. Pat. No. 5,422,252. This allows thestrand displacement reaction to generate many copies of newlysynthesized sequence, which form the basis of a non-thio stranddisplacement amplification as described in U.S. Pat. No. 6,191,267. Thenext step in the process is to use these amplified newly synthesizedfragments, complementary to the strand displacement primers as DNAbridges to generate a signal with the capture oligonucleotide, asdescribed above. This is illustrated in FIG. 11, where in the stranddisplacement assay the ssDNA 3′-OH moiety (308, 310) binds to a region320 at the 3′ end of 319 composed of regions 320, 321 and 322. Anextension reaction 323 then occurs which is then nicked in reaction 324to produce a short portion of ssDNA 325 which accumulates by virtue ofcycling reaction 326 of primer extensions and nicks. Detection ofelement 325 is achieved by binding a first portion of 325 tocomplementary 40 immobilized on bead 39 and a second portion of 325 to alabeled polynucleotide 317. The label is then recognized by an antibodybound to alkaline phosphatase 318.

Removal of Primers after Amplification

We describe several novel approaches to remove unused PCR primers fromcompleted PCR reactions. It has been found that a consequence of seekingto develop systems incorporating rapid PCR reactions, i.e. completedamplification in less than about minutes, that it is necessary toincrease the primer concentrations. However, this typically can generatean increased primer background in the detection step, which can reducesignal generation on the capture oligonucleotide. Experiments usingpurified amplicons and increased unlabelled target oligonucleotides,amongst labeled control oligonucleotides, demonstrated that thesebackground oligonucleotides were able to remove or reduce the signal.One approach or a combination of the approaches described below can beused to reduce the background signal.

One way for providing for easy removal of primers from the reactionamplification mixture is to use a clam-like oligonucleotide primer. Thisoligonucleotide predominantly exhibits a certain desired secondarystructure in solution, when in a first temperature range, but not in asecond higher temperature range. In this example, the oligonucleotide iscapable of priming the target nucleic acid in the second temperaturerange, but not in the first temperature range. This is achieved bydesigning the oligonucleotide such that the primary structure results ina secondary structure with one or more regions that hybridize,preferably predominantly in an intra-molecular manner, but also in aninter-molecular manner. This can occur in the first temperature rangebut not in said second temperature range, thus changing the temperaturewill enable switching the primer between a priming and non-priming form.As a result, lowering the temperature at the end of the amplificationreaction effectively removes excess primer from the mixture. It has beenfound that clam-like primers of this type may be prepared incorporatinga polymerase blocking region, a single stranded hybridization region andoptionally a detectable moiety. Alternative methods for removing primerat the end of the amplification reaction have also been devised. Theseare by electrophoresis, post-PCR hybridization and enzymatic conversion.

Electrophoretic Separation

The first approach described is electrophoretic separation. It is wellknown that nucleic acids can be separated based on their molecularweight. By exploiting the size differences between the PCR amplicon andthe oligonucleotide primers it is possible to rapidly purify theamplicon. In the preferred embodiment, an electrophoresis module isincorporated into a single-use device. For example, the electrophoreticpurification module can be situated at a point along a channel in thedevice at a position convenient to effect purification, as shown in FIG.12. The device is comprised of an electrode 50 in a channel of thedevice and a second electrode 51 in an adjacent cavity 52. Eachelectrode is connected to an electrical contact pad 53. A channel 54 inthe device provides a means through which fluid moves from an earlierstage e.g. a PCR amplification step, to a later stage e.g. a detectionstep.

The purification module shown in FIG. 12 can be situated on either sideof the channel and above or below. It can have two or more electrodes.For example, an additional third electrode can be situated in a positionbetween the two electrodes that are shown. For the two-electrodeembodiment shown in FIG. 12, a capture membrane for the primer sequencesis used which effectively irreversibly absorbs the primer. Suitablematerials include nitrocellulose. Whatman DE52 membrane, and other DNAbinding membranes, well known in the art.

In one embodiment, solidified gel matrix, e.g. agarose, with anelectrophoresis buffer is positioned in the cavity. A sample segment ofPCR amplified material is then moved through the channel and positionedover the cavity. Optionally a second pair of conductivity electrodes canbe used to sense the position of the material as it moves through thechannel, as described in jointly owned U.S. Pat. No. 5,096,669incorporated herein by reference. Once the sample is positionedappropriately, an electrophoretic charge is applied across the twoelectrodes, with 50 being negative, and 51 being positive. This causeselectrophoretic movement of the molecules in the gel matrix, with thesmaller synthetic oligonucleotide primers moving the fastest and thelarger per amplicons moving slower. Once the fragments have moved anappropriate distance, i.e. out of the channel and into the cavity, theelectrophoretic charge is reversed, causing the fragments to move in theopposite direction. After a certain amount of time and with a particularcharge and voltage the larger molecule will have transferred back intothe channel, leaving the smaller primer molecules in the gel material.This is thus a way of effecting purification of the amplicons.

In another embodiment, a third electrode is positioned between the twoelectrodes shown in FIG. 12. Here electrodes 50 and 51 are set asnegative and positive respectively. After a time when the primermolecules have passed the third middle electrode, but the arnplicon hasnot, electrode 50 is reversed to positive charge, leaving electrode 51as positive. At this point, the third middle electrode is made negative.This causes the primer to continue moving away from the channel, andreverses the direction of the amplicon back towards the channel.

FIG. 13 (a)-(g) illustrates the steps involved using charged dyes in adevice. FIG. 13( a) shows a modified i-STAT cartridge base of the typedescribed in jointly owned U.S. Pat. No. 5,096,669. It has an entry port71, a channel 72, a cavity 73 adjacent to the channel and threeelectrodes 74, 75 and 76, two of which are in the cavity and one in thechannel. The cavity contains 1% agarose with buffer as a transparentgel. A sample comprising 5 uL of common electrophoresis loading dyes,bromophenol blue and xylene cyanol, both negatively charged, is addedthrough the entry port and enters the channel as a fluid segment 77, asshown in FIG. 13( b). Note that these dyes migrate at roughly 25 to 50bp sizes, where as with actual DNA separation will be of 50 bp and 300bp fragment.

In FIG. 13( c) a negative potential is applied to 74 and a positive oneto 76, in this case 50V. The charged dyes quickly move into the agarosegel, towards 76. The two dyes migrate at different rates according totheir charge-to-mass ratio through gel. As shown in FIG. 13( d) the dyesare resolved into two bands 78 and 79 either side of 75. This takesabout three minutes. At this point 74 and 76 were made positive and 75made negative, thus driving the two migrating dyes in oppositedirections as shown in FIG. 13( e) until the xylene cyanol dye re-entersthe channel, FIG. 13(J). Finally, the xylene cyanol is pneumaticallymoved down the channel for further downstream applications, as shown inFIG. 13( g) while the other dye remains in the cavity.

Clearly, the behavior of the two dyes is representative of differentlength nucleotide sequences or any other chemical species with differentcharge-to-mass ratios that could be separated from one another quicklyusing electrophoresis. Furthermore, the electrophoretic properties andcapabilities of this device can be tailored according to gel density,buffer-salt selection, applied potential and duration, physicaldimensions and the like, to achieve any desired separation.

In another embodiment, the original liquid sample is moved out of theregion of the channel abutting the cavity and is replaced with a smalleramount of a different liquid prior to reversing the polarity of theelectrodes. This can effect a concentration of the amplicon, which inturn can increase hybridization rates at a later stage in the assayprocess. In another embodiment, the primers are brought in contact witha capture membrane or particle within the cavity, which effectsirreversible binding, thus preventing the primer from moving backtowards the channel. In another embodiment, the agarose may be replacedwith a different matrix including acrylamide, a mixture of agarose andlocust bean gum, hydrocolloids, or other appropriate separation media.In another embodiment, the device is manufactured as a subcomponent onsilicon and inserted into a micro-device, as shown in FIG. 12. Inanother embodiment, to address constraints associated with integrationof this separation component into a genetic testing device, theelectrophoretic channel may be L-shaped with electrode 75 located at ornear the elbow of the ‘L.’ For example FIG. 24 shows the L-shapedchannel feature 655 incorporated into an integrated testing device 651abutting conduit 409, with electrodes 652, 653 and 654 with entry port657 and matrix 656. Other elements are as for FIG. 19.

FIG. 14 demonstrates the operation of the electrophoresis device with anamplicon and primer from a PCR reaction. Lane (A) shows a portion of thePCR reaction product after electrophoresis into gel cavity and back outagain and into a fresh second recovery aliquot and applied to a 6%non-denaturing acrylamide gel. Lane (B) shows a portion of sample thatremained in original aliquot removed after one direction migration. Lane(C) is a control of equivalent concentration to the sample and lane (D)is a 10 base-pair ladder at a three times greater concentration than inthe sample and control. The ladder major species base-pair lengths are330, 100 and 10.

Clam-Like Oligonucleotides

Normally, for PCR applications reducing the amount of secondarystructure is a desirable approach when designing syntheticoligonucleotide sequences, as this helps in reducing non-specific andpoor priming of the target. The predicted folding structure of anoligonucleotide that is complementary to the Hfel gene, that has a fivebase pair adenoside spacer sequence and that has a free single strandedregion is shown in FIG. 15. The FIG. 15 sequence is5′-ACTTCATACACAACTCCCGCGTTGCATAACTAAA-AACTGGCAAGGGTAAACAGATCCCC-3′(SEQUENCE ID No. 5). As a theoretical prediction of potential molecularfolding an RNA folding program (Vienna RNA) predicts an oligonucleotidewith single stranded nature at any temperature above 10° C. By designingsynthetic oligonucleotides with secondary structure at low temperatures,but which lose their secondary structure during the denaturation step ofPCR and PCR hybridization, we can effect hybridization of amplicons butnot the primer molecules at the later stage of hybridization anddetection. Using the is Ol5 sequence as a starting point,oligonucleotides with a hairpin loop structure were designed and modeledas shown in FIGS. 16( a) and (b). The base pair sequence in FIG. 16( a)is 5′-TTGCCAGACTTCATACACAACTCCCGCGTTGCATAACTAAAAAGTATGAAGTCTGGCAAGGGTAAACAGATCCCC-3′ (SEQUENCE ID No. 6), and that 30 of FIG. 16(b) is5′-ACCCTTGCCAGACTTCATACCCGCGTTGCATAACTAAAAA-GTATGAAGTCTGGCAAGGGTAAACAGATCCCC-3′(SEQUENCE ID No. 7). In the models of FIG. 16, a five base pair sequenceshown in the box is used to model the effect of an HPEG spacer. Based onthe models in FIG. 16, two oligonucleotides designated CLAM1 and CLAM2were. The two sequences differ by four nucleotides.

CLAM1: 5′-TTGCCAGACTTCATACACAACTCCCGCGTTGCATAACT-HPEG-GTATGAAGTCTGGCAAGGGTAAACAGATCCCC-3′ CLAM2:5′ACCCTTGCCAGACTTCATACCCGCGTTGCATAACT-HPEG-GTATGAAGTCTGGCAAGGGTAAACAGATCCCC-3′

In the CLAM1 sequence, the sequence located 5′ to the HPEG spacer,5′-TrGCCAGACTTCATACACAACTCCCGCGTTGCATAACT-3′ is designated as SEQUENCEID No.8 and the sequence located 3′ to the HPEG spacer,5′-GTATGAAGTCTGGCAAGGGTAAACAGATCCCC-3′ is designated as SEQUENCE IDNo.9.

In the CLAM2 sequence, the sequence located 5′ to the HPEG spacer,5′-ACCCTTGCCAGACTTCATACCCGCGTIGCATAACT-3′ is designated as SEQUENCE IDNO. 10 and the sequence located 3′ to the HPEG spacer,5′-GTATGAAGTCTGGCAAGGGTAAACAGATCCCC-3′ is designated as SEQUENCE ID NO.11.

These oligonucleotide sequences maintain the key primary sequencefeatures for Hfel priming in PCR reactions and for binding to thecapture oligonucleotide, but additional sequences have been added togenerate intramolecular binding, generating these “clam-like”structures. Note that the HPEG spacer region sequence is indicated withthe five ‘A’s and it was anticipated that these sequences will have nosecondary structure above about 40° to 45° C.

FIGS. 7( b) and 7(c) compare and contrast the differences between usingPCR primer sequences with little or no secondary structure and the CLAMPCR primers. At temperatures during PCR, particularly at temperatures ator above hybridization, the CLAM primers do not form secondarystructures and once it is incorporated into a PCR amplicon it loses itsability to form a clam structure. At temperatures below PCRhybridization and at temperatures used for hybridization of the captureoligonucleotides, the CLAM PCR primers do form secondary structure.Therefore, unincorporated CLAM PCR primers do not bind to the captureoligonucleotides and do not interfere with signal generation.

FIG. 7( b) shows a PCR reaction using a non-CLAM oligonucleotidesequence and hybridizing to a target nucleotide sequence. A sequencelike is 015 with no secondary structure is used as one of two PCRprimers 81. The PEG spacer generates single stranded regions in the PCRamplicon and excess primer sequences are generated in the reaction 82.In step 83, both the PCR amplicon and the unreacted primer sequences canbind to the capture oligonucleotide bound to a solid substrate like abead. Typically, the unreacted primer is in significant molar excesscompared to the PCR amplicon and reduces the signal detection.

FIG. 7( c) shows a PCR reaction using a CLAM oligonucleotide sequencehybridizing only the PCR amplicon to a target nucleotide sequence. Usinga modification to the is O15 sequence to generate either CLAM1 or CLAM2sequences, a PCR reaction is performed 81. At temperatures used in PCR,the secondary structure is eliminated. Once one end of the CLAMoligonucleotide is incorporated into a PCR amplicon it no longerfunctions with the secondary structure and provides a single strandedregion 82. In step 83, the temperature is below that required togenerate secondary structure of unincorporated CLAM primer sequences. Asa result. CLAM primers that have been incorporated into a PCR ampliconwill have single stranded regions capable of binding to the captureoligonucleotide.

Enzymatic Removal

Two enzymatic approaches have been devised for removal of primers, theserelate to TdT-tails on unincorporated oligonucleotides and degradationof unincorporated oligonucleotides. Within a PCR reaction mixture thereexist two types of structures, amplicons with single stranded regions,in the example above having an iSpl8 primer and unincorporated syntheticoligonucleotides. The primers on the amplicons only have extending 5′regions, whereas the unincorporated primers have free 5′ and 3′ singlestranded ends. Using enzymes specific to these differences at the 3′end, strategies to differentially remove these molecules was developed.

Calf Thymus Terminal deoxynucleotidyl transferase (TdT) enzymatictreatment of the PCR reaction product is specific to single stranded 3′extensions, thus only the unincorporated primer will generate a newlyincorporated tail. By contrast, the amplicon only has single strandedregions with 5′ tails, which are unreactive with TdT.

While it is inefficient and not unique for a universal capture system,one could use a single nucleotide (dNTP) such as ‘T’ to create anextended T tail at the 3′ end of the PCR primer. Any nucleotide,including modified nucleotides, including ribonucleotides could be usedfor this application and which function with TdT or poly(A) polymerase.The modified PCR reaction mix with T tailed unincorporated primersequences can then be exposed to a capture oligonucleotide with apoly(A) sequence. Only unincorporated PCR primers with Ttails will bebound to the capture poly(A) sequence. This enriches the reactionmixture for PCR amplicons with associated poly(T) sequences. The poly(A)capture oligonucleotide can be bound to solid surfaces, beads, in amatrix like agarose, acrylamide, poly vinyl alcohol or other appropriatehydrocolloids.

An alternative method is based on the use of an endonuclease. As theunincorporated oligonucleotide primer has a free 3′-hydroxyl group andthe ampIicon does not, a 3′-5′ exonuclease is employed to removeunincorporated oligonucleotide primer. Enzymes including ExoI and ExoThave specific 3′-5′exonuclease activity with single stranded DNA withfree 3′-hydroxyl groups. In this embodiment it is preferable to useprimers with 5′phosphate groups.

Post-PCR Hybridization

In the PCR reaction described above, amplicons are generated containingtwo primers which generate two different single stranded regions. Inorder to generate a signal, both single stranded regions are necessary,as well as the newly amplified region, which is a bridge between the twosingle stranded regions.

In this example, single stranded A region binds to the complementary Aprime capture oligonucleotide at the biosensor. The single stranded Bregion binds to a synthetic oligonucleotide B-prime which has a moietyfor the enzymatic conjugate. Alternatively, the enzymatic conjugatebinds directly to the B region.

By first creating a solid substrate with B prime captureoligonucleotides bound to a solid substrate, and in this example in achannel leading to the detection region, and allowing the PCR reactionmaterial to hybridize under the appropriate conditions, any B regionoligonucleotides that were not incorporated into amplicons are lost fromthe channel, enriching the channel for B region oligonucleotides and Bregion oligonucleotides incorporated into amplicons. Unbound material iswashed away.

The enriched bound B region oligonucleotides and amplicons are thenreleased from the solid support by heat or alkaline conditions. Thematerial is allowed to move towards the detection region of the device.Oligonucleotides with A regions or oligonucleotides incorporated intoamplicons will be bound to A prime capture oligonucleotides at thebiosensor. The biosensor can be washed, removing any unincorporated Bprimers, leaving only fully incorporated amplicons. This effectivelyremoves background from unincorporated oligonucleotides.

Detailed Description of Nucleic Acid Testing Cartridges

An integrated single-use device for performing a nucleic acid analysisand its interaction with the reading instrument is shown topologicallyin FIG. 6. It comprises a housing 100 with an entry port 101 foraccepting a sample suspected of containing a target nucleic acid. Theentry port leads to a chamber 102 which has a reagent for extractingsaid target nucleic acid. The reagent 103 can be coated on to the wallof the chamber. The chamber may contain beads 104, e.g. magnetic beadswith a coating suitable for binding nucleic acid. The chamber alsopreferably contains a wax, which can melt to form a contiguous wax layer105 in the region of egress to a conduit 106. Once the preferredmagnetic beads have associated with said target nucleic acid a magneticfield is applied to draw them through the wax layer and into theconduit. Note that this applied magnetic field may also be oscillated inthe chamber to promote extraction of nucleic acid from the sample.Optionally a wash fluid may be applied to the beads prior to leaving theextraction chamber. A wash fluid chamber 122 is connected between theentry port and the extraction chamber. In addition, a sample and washfluid waste chamber 123 is connected at the distal end of the extractionchamber, with respect to the entry port. In operation, after theextraction step the beads are held on the wall of the chamber bymagnetic means and the wash fluid is then passed from chamber 122through chamber 102 and into chamber 123. This displaces unwanted samplematerial and leaves chamber 102 containing the beads and predominantlywash fluid. The instrument 200 contains an actuating means 211 which isaligned to chamber 122 and provides a force to a flexible diaphragm 124to expel the wash fluid out of the chamber.

After washing, the beads then pass through the wax layer and intoconduit 106 and then into the amplification chamber 107. Movement of thebeads in the conduit is preferably by the same magnetic means, or can bepneumatic. The amplification chamber is also attached to anamplification reagent holding chamber 108, which can deliver thesereagents to the amplification chamber with the beads, as in thepreferred embodiment, or in a separated step before or after the beadsenter this chamber. Alternatively, these reagents may reside in thischamber and element 108 omitted. In another alternative whereamplification reagents are best dry-stored, chamber 108 may containdiluents and the reagents coated onto the wall of the amplificationchamber.

The amplification reagents as described above can provide for variousamplification methodologies, e.g. rolling circle and ligase chainreaction. In the preferred embodiment the reagents incorporate adetectable moiety into an amplified target by means of PCR. Optionally,an applied magnetic field may be used to provide mixing of the beads inthe amplification chamber. This is in the same manner as described forthe extraction chamber.

The amplification chamber also has a heating element 109 and atemperature sensing thermistor 110 for controlling the temperature ofthe amplification chamber and thus effecting conditions suitable foramplification of the target nucleic acid. In the preferred embodimentthe amplification chamber is cycled between 680 C and 900 C for thirtycycles. The time duration at each temperature is more than 5 and lessthan 30 seconds respectively. While the main part of the housing 100 ismade of plastic, at least one wall of the amplification chamber is madeof an inert material with superior thermal conduction properties,preferably silicon. The reverse side of the silicon has a resistive path111 and two electrical contact pads 112 and 113 which constitute theheating element 109. An electric current passing through the resistivepath causes heating of the silicon chip and thus the contents of theamplification chamber. The reverse side of the silicon also has athermistor 110 wired by leads 114 to two electrical contact pads 115 and116. The output of the thermistor is used by the instrument to controlthe current passing through the resistive path and thus the temperatureof the amplification chamber.

The single-use device 100 may also optionally include closure element117 to seal the entry port. This can be a plastic snap-closure elementof the type described in jointly owned U.S. Pat. No. 5,096,669 or theslide closure of jointly owned pending U.S. application Ser. No.10/658,528.

The amplification chamber may also be sealed at the ingress and egressby 118 and 119 respectively. This is desirable for ensuring reagentsremain in the chamber during temperature cycling. For example, element118 and 119 may be deformable rubber seals. Actuation can be by pinelements 209 and 210 in the instrument, which move through opening 120and 121 in the housing to contact 118 and 119 and cause sealing. Pinelements 209 and 210 may be actuated independently or together by theinstrument.

The egress of the amplification chamber is attached to a second conduit125 containing a sensing region 126 comprising an immobilized captureoligonucleotide 127 and a sensor 128. The housing 100 contains a secondpump means 129 attached to the amplification chamber for moving theamplified target to said sensing region. The pump means comprising anair-filled chamber 130 with a diaphragm 131. The instrument 200 containsan actuating means 212 for applying a force to element 131 topneumatically displace air from chamber 130 and thus displace theamplified target towards the sensing region.

When the amplified target arrives in the detector region it can bind tothe capture oligonucleotide and be retained. The detection region alsocontains a dry reagent layer coated onto the wall 151. In the preferredembodiment, the moiety associated with the primer (which becomes part ofthe amplicon) is biotin and the dry reagent 151 is streptavidin-labeledalkaline phosphatase. Dissolution of the reagent with the ampliconcauses it to bind to the biotin via the well known biotin-avidininteraction. In operation this step generally takes from about 5 toabout 15 minutes. In alternative embodiments the moiety can be 5′ FAM or15 5′-biotin and the dry reagent anti-FITC-ALP (alkaline phosphatase) orstreptavidin-glucose oxidase conjugate.

A third conduit 132 is attached to the second conduit 125 between theegress of the amplification chamber and the sensing region. It has achamber 133 with a detection reagent 134. Optionally, the reagent iscontained in a flexible sealed foil pouch 135 and in operation theinstrument contains an actuating means 213 which can provide force tothe pouch and cause it to rupture by being pressed against a rupturingfeature 136, preferably a sharp plastic point molded into the housing.This caused the detection reagent to move out through the third conduitand into the second conduit. This displaces and washes away anyuncaptured amplified target and other material from the sensing regionwhile permitting amplified target to remain bound to the captureoligonucleotide. The housing 200 also contains a waste chamber 137attached to the second conduit for receiving the displaced material.

In the final step, the detection reagent reacts with the moiety 138incorporated into said amplified target 139 to generate a signal at thesensor 140. In the preferred embodiment where the moiety is biotin andis bound to streptavidin-labeled alkaline phosphatase, the detectionreagent is p-aminophenol phosphate which is hydrolysed to formp-aminophenol by the enzyme. This is then electrochemically oxidized atthe electrode surface of an amperometric sensor to generate a currentproportional to the amount of moiety that is present, as illustrated infigures showing chronoamperometry (current versus time plots).

The instrument, 200 in FIGS. 6 and 650 in FIG. 21, used to operate theintegrated single use device is shown interacting with the test devicein FIG. 21. It includes a port 654 for receiving the single-use device100 and 651. The instrument has a keypad 652 for user entries and adisplay 653. One or more locating features 202 for locating the devicewith respect to the instrument to provide for the desired interaction ofelectrical connecting elements and actuating elements are provided. Theinstrument contains an electromagnet 203 adjacent to the location of thebeads 104 in chamber 103. The electromagnet may be used to move thebeads from the extraction chamber to the amplification chamber and topromote mixing of the beads within each chamber. The instrument includesan actuating means 204 adjacent to the location of the amplificationreagent holding chamber 108 which can provide pressure to the chamberand cause the reagent to be displaced into the amplification chamber.The instrument also has a pair of electrical contacts 205 and 206 forcontacting element 112 and 113 and a power source for passing a currentthrough 111. It also includes a pair of electrical contacts 207 and 208for contacting element 115 and 116 for contacting the thermistor 110.Furthermore, the instrument includes suitable electrical circuitry andan embedded algorithm for controlling the temperature of theamplification chamber through these means.

The instrument includes actuation pin elements 209 and 210, which movethrough opening 120 and 121 in the housing to contact and close 118 and119 to seal the amplification chamber. Suitable electromechanicalfeatures are included to effect this actuation along with a controllingalgorithm for initiating sealing at the appropriate step in the analysiscycle.

The instrument also has an electrical connector of the type described injointly owned It is used to make electrical connection to the sensor 128in the housing 100. Where it is desirable to perform the detection stepat a controlled temperature, e.g. 37° C., the connector alsoincorporates heating and thermistor elements, which contact the backside of the silicon chip that provides the substrate for the sensor.These elements are of the same type as described for the amplificationchamber. The instrument has amperometric circuitry for controlling thepotential of the sensor and measuring current. The instrument also hasan embedded algorithm for controlling the entire analysis sequenceperformed by the instrument on the single-use device to make a nucleicacid determination and display a result on a display screen on theinstrument. Where the electroactive species generated or consumed inproportion to the captured target is more appropriately detected bymeans of potentiometry or conductimetry, alternative circuitry wellknown in the art is incorporated into the instrument.

In an alternative embodiment, the single-use device is composed of twoseparate parts as shown in FIGS. 19 and 20. FIG. 19 illustrates aseparate extraction device 470 and a combined amplification anddetection device 471. The elements in a combined form have the samefeatures as those shown for the integrated device in FIG. 6, with theexception of features related to transferring extracted material fromone to the other. Element 470 comprises an entry port 413, conduit 411,wash fluid 417 and waste chambers 418, a separation region 421, aterminal portion of the conduit 601 and an egress port 502 which mateswith ingress port 502. It also has mating features 520 and 521 whichmatch one or more opening 500 in 471. Element 471 has an amplificationchamber 410, conduit 409, chambers 408,409 and sensors 419, 420, exitconduit 405 and sealing feature 406. FIG. 20 is similar to FIG. 19, withthe difference that it comprises a combined extraction and amplificationcomponent 472 and a separated detection component 473. The matingfeatures are appropriately located between the two.

FIG. 18 shows an additional embodiment where a filter region 421 isintegrated into a device that performs extraction, amplification anddetection. Other elements are as for FIG. 19. FIG. 17( a) shows anoptical detection-based single-use cartridge where an optical sensor isintegrated into the device that is interrogated by a reflectance method.Light is generated by element 401 and interacts with sensor 403 and iscaptured by detector 400. FIG. 17( b) shows an optical single-usecartridge where the sensing region is a cuvette feature 404, permittingdetection with a light source 402 and detector 400 integrated into theinstrument.

It has been found that where the sample is a buccal swab, the extractioncomponent element, either magnetic or filter based, is unnecessary andthe sample may be directly inserted into the amplification chamber. FIG.28( a) and FIG. 28( b) show two views (top and bottom) of a buccalsample device for direct application of a buccal sample to a perchamber. This extraction and amplification device attaches to thedetection cartridge, by means of the mating features described above(not shown).

The general dimensions of the housing 100 are about 6 cm in length, 3 cmin width and 0.3 cm in height. The conduits and other features arepreferably rendered in a device base 143 and a device cover 144 whichare held together by an intervening double-sided adhesive tape 145, seeFIG. 6. Where the base and cover are injection molded in plastic,typically ABS or polycarbonate, conduits and recesses to accommodatesilicon chips, fluid containing pouched and the like are moldedfeatures. In this embodiment the adhesive tape acts as a sealing gasketto confine liquids to the desired conduits and chambers. Detaileddiscussion of the use of molded cover and base elements along with theuse of adhesive tape gaskets is found in jointly owned U.S. Pat. No.5,096,669 and pending US 20030170881 which are incorporated here byreference.

Detailed Description of Detection

The preferred method of detection in the single-use cartridge iselectrochemical, however other sensing methods including fluorescence,luminescence, colorimetric, thermometric, fiber optics, optical waveguides, surface acoustic wave, evanescent wave, plasmon resonance andthe like can be used.

The preferred sensor 128 comprises an amperometric electrode 300, whichis operated with a counter-reference electrode 301 and is shown in FIG.6. The amperometric electrode 300 comprises a 100 um diameter gold layermicrofabricated onto a silicon chip 302. The silicon chip is treated inthe first step of manufacture to produce an insulating layer of silicondioxide on the surface, as is well known in the art. The electrode isconnected by means of a conducting line 303 to a connector pad 304 whichmakes contact with the electrical connector of the instrument. Theconducting line is typically coated with an insulating layer ofpolyimide 305. Directly over the electrode 300 or at an adjacentlocation 306 on the chip are adhered polymer particles 307 that have aligand 308 complimentary to and capable of capturing the amplifiedtarget. The counter-reference electrode may be microfabricated on thesame silicon chip or one place adjacently in the second conduit 125. Itcomprises a silver-silver chloride layer, of 200 um diameter attached bya contact line 309 to a contact pad 310 that makes contact with theinstrument connector. Again the line 309 is preferably coated with aninsulating layer of polyimide. A detailed description of amperometricsensor microfabrication is found in jointly owned U.S. Pat. No.5,200,051 which is incorporated here by reference.

A conductivity sensor comprising two conductive bars 311 and 312 arepresent on chip 302, or an adjacent chip 350, connected to contact pads313 and 314 by lines 315 and 316 respectively, see FIG. 6. Theconductivity sensor can be used by the instrument to distinguish ifliquid or air is in contact with the sensor and thus determine theposition of a solution in the second conduit with respect to the sensor300. This solution may be one containing the amplified target or thedetection reagent. Optionally a conductivity sensor may be incorporatedinto or adjacent to both the extraction chamber and the amplificationchamber to determine the position of a fluid. A detailed description ofconductivity sensor microfabrication and use is found in jointly ownedU.S. Pat. No. 5,447,440 and U.S. Pat. No. 6,750,053 which areincorporated here by reference.

In an alternative embodiment of the single-use device 100 a transparentglass window is substituted for the silicon chip 302 and the sensingregion of the device forms a cuvette, FIG. 17. The amplified targetcapture reagent is immobilized on the glass and in this case thedetection reagent contains a molecule that the moiety, e.g. alkalinephosphatase, causes to generate an optically detectable signal, e.g.fluorescence. Such molecules are well known in the art. In all otherrespects the operation of the single-use device is the same as in theelectrochemical detection mode.

Detailed Description of Nucleic Acid Testing Cycle with Single-UseDevice

The preferred embodiment of an assay cycle using the single-use device100 in conjunction with the instrument 200 is as follows. Anapproximately 10 uL blood sample is added to the entry port 101 and isdrawn by capillary action into the extraction chamber 102. An entry portclosure element 117 is then used to seal the entry port. Reagents 103comprising a chaotropic agent, lithium dodecylsulfate and dithiothreitoland a chelating agent, ethylene diamine tetraacetic acid, which arecoated on the wall of the chamber dissolve into the blood sample andcause lysis of the cells and permit nucleic acid from within the cellsto be liberated and to be adsorbed onto the carboxylate coating on themagnetic beads 104. A magnetic field can be used to agitate the beads topromote mixing within the chamber and speed up the rate of extraction.This step of the extraction process generally takes about 0.3 to lessthan 1 minute. Where the magnetic field is deployed, this is under theautomatic control of the instrument and is determined by an embeddedalgorithm that controls the test cycle. Once this step is complete, theinstrument deploys a magnetic field which holds the magnetic particlesto the side of the extraction chamber. Wash fluid from the wash fluidchamber 122 is then pneumatically forced into the extraction chamber andflushes the contents into the wash fluid waste chamber 123. Note thatthe wash fluid waste chamber has a vent 146 and that during this stepthe instrument seals the ingress 118 to the amplification chamber, thuswaste fluid is directed into the waste chamber rather than enteringconduit 106. This step takes about 30 seconds. The wash fluid in thepreferred embodiment is deionized water and the volume of wash fluidthat passes through the extraction chamber is 20 to 30 uL. Note alsothat the silicon chip that forms one wall of the amplification chamberalso forms one wall of the extraction chamber, as shown in FIG. 23, thusthe extraction process can be performed at a controlled temperature. Inthe preferred embodiment nucleic acid extraction from blood occurs atroom temperature.

In the next step, the instrument opens the ingress seal 118 and releasesthe magnetic particles from the wall of the extraction chamber and drawsthem through the wax layer at the boundary of the extraction chamber andconduit leading to the amplification chamber. The instrument ensuresthat the temperature of the extraction chamber is sufficient for the waxto be in liquid form and permit the magnetic particles to pass through.In the preferred embodiment the wax is paraffin and the controlledtemperature is at between 45 to 700 C. As discussed previously passageof the particles through the wax minimizes interferents of PCRamplification, which can include hemoglobin. The particles are thendrawn into the amplification chamber. In the preferred embodiment theamplification chamber has a volume 10 of 10 to 20 uL. As shown in FIG.23 the chamber 606 is “U” shaped having a total length of 8 mm, width of8 mm and height of 0.25 mm Other features of the element 609 shown inFIG. 23 are chambers 600 and 602, ports 603, 604 and 607, conduits 601and 608, and heater 605.

The next step of the process involves the instrument pneumaticallydisplacing the PCR amplification reagent from its chamber into theamplification chamber. The PCR amplification reagents comprise DNApolymerase, a buffer and a modified primer. The primer comprises asequence of bases complimentary to a first region of the target nucleicacid, a polymerase blocking region, a single stranded hybridizationregion attached to the polymerase blocking region with an attacheddetectable moiety, which is biotin. In the preferred embodiment thebuffer consists of 22 U/ml Thermococcus species KOD, thermostablepolymerase complexed with anti-KOD antibodies. 66 mM Tris-S04 (pH 8.4),30.8 mM (NH4)2S04, 11 mM KCL. 1.1 mM MgS04, 330 uM dNTPs, as well asproteins and stabilizers (Invitrogen Life Technologies AccuPrime PfxSuperMix manual, Cat. No. 12344040), but alternatively could be 20 mMTris-HCL (pH 8.8), 2 mM MgSO4, 10 mM KCI, 10 25 mM (NH4)2S04, 0.1%Triton-X-100, 0.1 mg/ml nuclease-free BSA as described in the StratagenPfu DNA polymerase Instruction Manual Cat#600135 Revision$064003d).

In the next step the instrument seals the two sealing elements in thedevice, 118 and 119, to retain the beads and reagent in theamplification chamber and the cycles the temperature thirty timesbetween 95° C. and 99° C., and a hybridization step at 68 C withdurations at each temperature of 2 seconds and 12 seconds respectively.The overall amplification time is about 12 minutes. Once this step iscompleted, the amplified target is then transferred from theamplification chamber and into the conduit that leads to the detectionregion of the device. In one embodiment, at the end of the PCR reactiongaskets sealing the PCR chip entry and exit ports are lifted off of boththe entry and exit ports. An air bladder is depressed in the cartridge,creating a positive air pressure in the entry port gasket, forcing theliquid out of the exit port gasket, moving the liquid towards the finaldetection region of the chip. Here, a set of conductivity bars are usedfor monitoring the movement of liquid to the detection region.

In the preferred embodiment the clam-like primers are used, thus in theunheated conduit that leads to the detection region, these primersre-anneal to themselves and are effectively removed from the assay asinterferents. In an alternative embodiment, where electrophoresis isused to separate out unwanted primer the elements described in FIG. 12and FIG. 13 are combined into the single-use device as shown in FIG. 24.This separation process is described above. In the single-use devicewith electrophoretic separation, the instrument makes electricalconnection to the electrophoresis electrodes 74, 75 and 76 (see FIG.13), and 652, 653 and 654 (see FIG. 24). In the device the time for thisstep is typically less than 1 to 2 minutes, depending on the sizes ofprimer and amplicon. In another alternative embodiment where enzymaticremoval of unused primer is employed, the enzymatic mixture is appliedto a portion of the wall 150 of the conduit leading from theamplification chamber to the detection region. This material dissolvesonto the liquid containing the amplicon and converts the primer to anon-interfering form as described above. The dry reagent mixture on thewall is preferably the enzyme in a support matrix comprising trehaloseor ficoll, which promotes rapid dissolution. The time taken for theenzymatic step is typically about six minutes and is dependent on theamount of enzyme, temperature, type of primer being removed. In anotherembodiment, post-hybridization of the amplicons with a first captureoligonucleotide, which removes the detection region of the amplicons,followed by a wash step to remove any unbound unincorporatedoligonucleotides which would be involved in the final capture step canbe used. The amplicons and primers bound in the first capture step arethen un-bound using heat or alkaline conditions, then allowed to move tothe final detection region, where the capture oligonucleotides capturefully created amplicons.

In the next step the amplicon arrives in the detection region and thedissolution of the reagent on the wall of the detection chamber 151occurs. In the preferred embodiment this reagent is streptavidin-labeledalkaline phosphatase which binds to the moiety on the amplicon which ispreferably biotin to form a complex of amplicon and the enzyme. Thiscomplex can then bind to the capture oligonucleotide on the sensor.Depending on the kinetics the amplicon may also bind first to thecapture oligonucleotide and then the labeled enzyme. In the device thetime for this step is typically about 5 to 15 minutes.

In the final step detection reagent is displaced from the detectionreagent chamber into the sensing region, thereby displacing any unboundamplicon and labeled enzyme to the waste chamber. Elements 152 and 153which are constriction that cause turbulence in the region of the sensormay optionally be included to enhance the efficiency of thehybridization step, thus reducing the hybridization time and the amountof wash fluid that is required. In the device the time for this step istypically less than 70 seconds and the amount of wash fluid that is usedis about 10 to 50 uL. As stated previously the wash fluid also containsa reagent that enables detection. A trailing portion of the fluid isretained over the sensor, thus enabling the captured alkalinephosphatase to convert the reagent p-aminophenol phosphate top-aminophenol which is then oxidized at the electrode to give rise to ameasurable current. In the device the time for this step is typicallyless than 1 minute. Positioning of the trailing edge with respect to thesensor may be achieved using a pair of electrodes 155 and 156 forming aconductivity sensor as described above.

The measured current is used by the instrument to determine the presenceor absence of the suspected target nucleic acid in the original sample.This may be a qualitative result, or where the target is present, aquantitative determination of the amount of target in the sample. Analgorithm for a particular target factors the original sample volumeentering the extraction chamber, the number and efficiency ofamplification cycles and the efficiency of the capture reaction alongwith any other necessary factors to determine the original concentrationof the target in the sample. Such factors are independently determinedusing known samples from a reference method. These methods are wellknown in the art.

In a related embodiment, a second sensor 154 is provided in thedetection region to account for any non-specific binding of thestreptavidin-labeled alkaline phosphatase to the first sensor. Thesecond sensor is the same as the first but, has a captureoligonucleotide that does not bind to the amplicon. Any signal at thesecond sensor is subtracted from the signal at the first by thealgorithm. The overall time for the assay, from sample entry into thesingle-use device and insertion into the instrument, takes between about10 and 20 minutes and generally depends on the specific target and therequired number of amplification cycles. When the genetic test iscomplete and result is displayed by the instrument, the actuationmechanism within the instrument then releases the device and it can beremoved and discarded by the user. The instrument is then ready toreceive a new single-use device. A significant advantage of thedisclosed device and instrument combination is that once the sample hasentered the device, all other steps are controlled by the instrument,thus eliminating possible human-error in the test cycle. This means thesystem can be used reliably by those not specifically skilled inanalytical laboratory measurement. For example a physician may use thesystem at the bedside or during a patient's office visit. The system mayalso be used at remote locations, for example in environmentalmonitoring and hazard assessment. An added benefit of the design is thatit also retains sample residue and amplified material within the devicefor safer disposal.

In an alternative embodiment of housing 100, the extraction chamber 102contains a filter material 157 and 421, impregnated with extractionreagents comprising a chelating agent and a chaotropic agent. One wallof the extraction chamber is also composed of heating element with athermistor for controlling temperature. The filter material ispreferably composed of 3 MM Whatman paper and has a carboxylated surfacewhich preferentially binds nucleic acid. When the sample, e.g. blood,enters the extraction chamber, it dissolves the extraction reagent andnucleic acid from the cellular material binds to the filter. This stepof the extraction process takes about 0.5 to 2 minutes. A bolus of washfluid from the wash fluid chamber 122 is then pushed through theextraction chamber and exits into the wash fluid waste chamber 123,carrying away lysed cellular debris from the sample, while leaving theextracted nucleic acid adsorbed onto the filter. Multiple boluses ofwash fluid may be used to ensure a complete wash. A further bolus ofwash fluid is then pushed into the chamber and the instrument activatesthe heating element and controls the temperature of the bolus of fluidto 90° C., by means of the thermistor. This caused the nucleic acidabsorbed onto the filter to desorb from the filter and dissolve in thefluid. The fluid containing the nucleic acid material is thenpneumatically transferred to the amplification chamber. In thisembodiment the wash fluid is preferably deionized water.

<200>SEQUENCE CHARACTERISTICS: <210>SEQ ID NO: 1 <211>LENGTH: 31<212>TYPE: DNA <213>ORGANISM: Artificial  <220>FEATURE: <221>NAME/KEY: Misc_feature  <222>LOCATION: 1-31 <223>OTHER INFORMATION: sequence is synthesized  <400>SEQUENCE: 1ACTTCATACA CAACTCCCGC GTTGCATAACT <200>SEQUENCE CHARACTERISTICS:<210>SEQ ID NO: 2 <211>LENGTH: 20 <212>TYPE: DNA<213>ORGANISM: Artificial <220>FEATURE:  <221>NAME/KEY: Misc_feature <222>LOCATION: 1-20  <223>OTHER INFORMATION: sequence is synthesized <400>SEQUENCE: 2 TGGCAAGGG TAAACAGATC <200>SEQUENCE CHARACTERISTICS:<210>SEQ ID NO: 3 <211>LENGTH: 22 <212>TYPE: DNA<213>ORGANISM: Artificial <220>FEATURE:  <221>NAME/KEY: Misc_feature <222>LOCATION: 1-22  <223>OTHER INFORMATION: sequence is synthesized <400>SEQUENCE: 3 AACAATACCA CCGTAGCGAT CA<200>SEQUENCE CHARACTERISTICS: <210>SEQ ID NO: 4 <211>LENGTH: 22<212>TYPE: DNA <213>ORGANISM: Artificial <220>FEATURE: <221>NAME/KEY: Misc_feature  <222>LOCATION: 1-22 <223>OTHER INFORMATION: sequence is synthesized  <400>SEQUENCE: 4AACAATACCACCGTAGCGATCA <200>SEQUENCE CHARACTERISTICS: <210>SEQ ID NO: 5<211>LENGTH: 59 <212>TYPE: DNA <213>ORGANISM: Artificial <220>FEATURE: <221>NAME/KEY: Misc_feature  <222>LOCATION: 1-59 <223>OTHER INFORMATION: sequence is synthesized  <400>SEQUENCE: 5ACTCATACA CAACTCCCGC GTTGCATAAC TAAAAACTGG CAAGGGTAAA CAGATCCCC<200>SEQUENCE CHARACTERISTICS: <210>SEQ ID NO: 6 <211>LENGTH: 75<212>TYPE: DNA <213>ORGANISM: Artificial <220>FEATURE: <221>NAME/KEY: Misc_feature  <222>LOCATION: 1-75 <223>OTHER INFORMATION: sequence is synthesized  <400>SEQUENCE: 6TTGCCAGACT TCATACACAA CTCCCGCGT GCATAACTAAAAAGTATGAA GTCTGGCAAG GGTAAACAGA TCCCC <200>SEQUENCE CHARACTERISTICS:<210>SEQ ID NO: 7 <211>LENGTH: 79 <212>TYPE: DNA<213>ORGANISM: Artificial <220>FEATURE:  <221>NAME/KEY: Misc_feature <222>LOCATION: 1-79  <223>OTHER INFORMATION: sequence is synthesized <400>SEQUENCE: 7 ACCCTTGCCA GACTTCATAC ACAACTCCCG CGTTGCATAACTAAAAAGTA TGAAGTCTGG CAAGGGTAAA CAGATCCCC<200>SEQUENCE CHARACTERISTICS: <210>SEQ ID NO: 8 <211>LENGTH: 38<212>TYPE: DNA <213>ORGANISM: Artificial <220>FEATURE: <221>NAME/KEY: Misc_feature  <222>LOCATION: 1-38 <223>OTHER INFORMATION: sequence is synthesized  <400>SEQUENCE: 8TTGCCAGACTTCATACACAACTCCCGCGTrGCATAACT <200>SEQUENCE CHARACTERISTICS:<210>SEQ ID NO: 9 <211>LENGTH: 33 <212>TYPE: DNA<213>ORGANISM: Artificial <220>FEATURE:  <221>NAME/KEY: Misc_feature <222>LOCATION: 1-33  <223>OTHER INFORMATION: sequence is synthesized <400>SEQUENCE: 9 GTATGAAGTCTGGCAAGGGTAAACAGATCCCC<200>SEQUENCE CHARACTERISTICS: <210>SEQ ID NO: 10 <211>LENGTH: 35<212>TYPE: DNA <213>ORGANISM: Artificial <220>FEATURE: <221>NAME/KEY: Misc_feature  <222>LOCATION: 1-35 <223>OTHER INFORMATION: sequence is synthesized  <400>SEQUENCE: 10ACCCTGCCAGACTTCATACCCGCGTTGCATAACT <200>SEQUENCE CHARACTERISTICS:<210>SEQ ID NO: 11 <211>LENGTH: 32 <212>TYPE: DNA<213>ORGANISM: Artificial <220>FEATURE:  <221>NAME/KEY: Misc_feature <222>LOCATION: 1-32  <223>OTHER INFORMATION: sequenceis synthesized <400>SEQUENCE: 11 GTATGAAGTCTGGCAAGGGTAAACAGATCCCC

The above-described exemplary embodiments are intended to beillustrative in all respects, rather than restrictive, of the presentinvention. Thus, the present invention is capable of implementation inmany variations and modifications that can be derived from thedescription herein by a person skilled in the art. All such variationsand modifications are considered to be within the scope and spirit ofthe present invention as defined by the following claims

What is claimed is:
 1. A method for performing a nucleic acidamplification assay comprising: (a) combining reagents, DNA polymerase,a target nucleic acid and an amount of a first modified primercomprising a sequence of bases complimentary to a first region of thetarget nucleic acid, a polymerase blocking region attached to the firstmodified primer, a first single stranded hybridization region attachedto the polymerase blocking region, and a detectable label, (b) cycling amixture of (a) to provide multiple copies of an amplicon incorporatingthe first modified primer; (c) substantially eliminating excessunincorporated first modified primer from the mixture; (d) exposing themixture to a capture oligonucleotide complimentary to the first singlestranded hybridization region; (e) hybridizing the first single strandedhybridization region of the amplicon incorporating the first modifiedprimer, with the capture oligonucleotide; and (f) detecting the labelassociated with the hybridization.
 2. The method of claim 1, wherein:the combining further comprises combining an amount of a second modifiedprimer comprising a sequence of bases complimentary to a second regionof the target nucleic acid, a polymerase blocking region attached to thesecond modified primer, a second single stranded hybridization regionattached to the polymerase blocking region, and a detectable label; thecycling further comprises cycling a mixture of (a) to provide multiplecopies of an amplicon incorporating the first modified primer and thesecond modified primer; the substantially eliminating further comprisesthe substantially eliminating excess unincorporated second modifiedprimer from the mixture; exposing the mixture further comprises exposingthe mixture to a capture oligonucleotide complimentary to both of thefirst single stranded hybridization region and the second singlestranded hybridization region; and hybridizing further compriseshybridizing both of the first single stranded hybridization region andthe second single stranded hybridization region of the ampliconincorporating the first modified primer and the second modified primer,with the capture oligonucleotide.
 3. The method of claim 1, wherein thepolymerase blocking region is phosphoramidite18-O-dimethoxyltritylhexaethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.
 4. The method ofclaim 1, wherein the detectable label is biotin, FAM, or fluorocein. 5.A method for performing a nucleic acid amplification assay comprising:(a) combining reagents, DNA polymerase, a target nucleic acid and twomodified primers, the first modified primer comprising a sequence ofbases complimentary to a first region of the target nucleic acid, apolymerase blocking region, and a first single stranded hybridizationregion attached to the polymerase blocking region; the second modifiedprimer comprising a sequence of bases complimentary to a second regionof the target nucleic acid, a polymerase blocking region, and a secondsingle stranded hybridization region attached to the polymerase blockingregion; and where a detectable label is attached to at least one of thefirst and second modified primers; (b) cycling a mixture of (a) toprovide multiple copies of an amplicon incorporating the modifiedprimers; (c) substantially eliminating unincorporated modified primerwith the detectable label from the mixture; (d) exposing the mixture toa capture oligonucleotide complimentary to at least one of the first andsecond single stranded hybridization regions; (e) hybridizing the singlestranded hybridization region of the amplicon incorporating the modifiedprimer with the capture oligonucleotide; and (f) detecting the labelassociated with the hybridization.
 6. The method of claim 5, furthercomprising: receiving a sample comprising the target nucleic acid intoan integrated single-use device for performing the nucleic acidamplification assay; extracting the target nucleic acid from the sampleonto magnetic beads in an extraction chamber of the device; passing thetarget nucleic acid on the magnetic beads through a contiguous wax layerinto a first conduit of the device by means of an applied magneticfield; and displacing the target nucleic acid on the magnetic beads fromthe first conduit into an amplification chamber.
 7. The method of claim6, wherein the combining comprises pneumatically displacing thereagents, the DNA polymerase, and the two modified primers from achamber into the amplification chamber.
 8. The method of claim 7,further comprising melting a wax to form the contiguous wax layer in aregion of egress of the extraction chamber.
 9. The method of claim 8,further comprising: sealing sealing elements in the device to retain themixture and the magnetic beads in the amplification chamber; andtransferring amplified target nucleic acid from the amplificationchamber into a second conduit that leads to a detection region of thedevice.
 10. The method of claim 9, wherein the modified primersre-anneal to themselves in the second conduit and are effectivelyremoved from the nucleic acid amplification assay.
 11. The method ofclaim 6, wherein the polymerase blocking region for the modified primersis phosphoramidite 18-O-dimethoxyltritylhexaethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.
 12. The method ofclaim 6, wherein the detectable labels are biotin. FAM, or fluorocein.13. A method for performing a nucleic acid amplification assaycomprising; (a) combining reagents, DNA polymerase, a target nucleicacid and two modified primers, the first modified primer comprising asequence of bases complimentary to a first region of the target nucleicacid, a polymerase blocking region, a first single strandedhybridization region attached to the polymerase blocking region, and anattached detectable label; and the second modified primer comprising asequence of bases complimentary to a second region of the target nucleicacid, a polymerase blocking, a second single stranded hybridizationregion attached to the polymerase blocking region, and an attacheddetectable label; (b) cycling a mixture of (a) to provide multiplecopies of an amplicon incorporating the modified primers; (c)substantially eliminating unincorporated modified primers from themixture; (d) exposing the mixture to a capture oligonucleotidecomplimentary to at least one of the first and second single strandedhybridization regions; (e) hybridizing the single stranded hybridizationregion of the amplicon incorporating the modified primer with thecapture oligonucleotide; and (f) detecting labels associated with thehybridization.
 14. The method of claim 13, further comprising: receivinga sample comprising the target nucleic acid into an integratedsingle-use device for performing the nucleic acid amplification assay;extracting the target nucleic acid from the sample onto magnetic beadsin an extraction chamber of the device; passing the target nucleic acidon the magnetic beads through a contiguous wax layer into a firstconduit of the device by means of an applied magnetic field; anddisplacing the target nucleic acid on the magnetic beads from the firstconduit into an amplification chamber.
 15. The method of claim 14,wherein the combining comprises pneumatically displacing the reagents,the DNA polymerase, and the two modified primers from a chamber into theamplification chamber.
 16. The method of claim 15, further comprisingmelting a wax to form the contiguous wax layer in a region of egress ofthe extraction chamber.
 17. The method of claim 16, further comprising:sealing sealing elements in the device to retain the mixture and themagnetic beads in the amplification chamber, and transferring amplifiedtarget nucleic acid from the amplification chamber into a second conduitthat leads to a detection region of the device.
 18. The method of claim17, wherein the modified primers re-anneal to themselves in the secondconduit and are effectively removed from the nucleic acid amplificationassay.
 19. The method of claim 13, wherein the polymerase blockingregion for the modified primers is phosphoramidite18-O-dimethoxyltritylhexaethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite.
 20. The method ofclaim 13, wherein the detectable labels are biotin, FAM, or fluorocein.